Intracellular and extracellular blood magnesium fractions in hemodialysis patients; is the ionized fraction a measure of magnesium excess?

¹ Academic Medical Center, University of Amsterdam, Departments of Clinical Chemistry and
² Nephrology, F1–217, P.O. Box 22700, Amsterdam, 1100 DE, The Netherlands.

³ The work reported here was performed by Renata Sanders, Marjolein G. Klous, Henk J. Huijgen, Rudolf W. van Olden, and Gerard T.B. Sanders, with grateful appreciation for the technical assistance of Ron Hoebe and Carel van Oven of the Department of Radiobiology of the University of Amsterdam. Address correspondence about the Appendix to R.S. Fax 31-20-5664440.
^a Author for correspondence. Fax 31-20-5664440; e-mail h.huijgen{at}amc.uva.nl.

	Abstract

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To establish the best measure for determining magnesium overload, we measured ionized and total magnesium in serum and mononuclear blood cells and total magnesium in erythrocytes in blood of 23 hemodialysis patients, known for their disturbed magnesium homeostasis. When comparing the mean magnesium values obtained in the patient population with those of a control population, all of these magnesium markers, including the biologically active fractions, were significantly (P <0.05) increased. Because serum total magnesium was not increased in all dialysis patients studied, the population was divided into two groups, according to total serum magnesium >1.0 mmol/L or less than that. Results in these two populations showed that ionized serum magnesium and ionized magnesium in mononuclear blood cells might give a better indication about the magnesium status of the tested patients than the currently used total serum magnesium data. However, neither of the two markers, especially ionized serum magnesium, was able to discriminate fully between normal magnesium homeostasis and magnesium excess. We therefore conclude that the two ionized magnesium markers offer minimal advantage for this discrimination, and that the total magnesium concentration in serum remains the measurement of choice.

	Introduction

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In trying to obtain information about the magnesium (Mg) status of a patient, clinician and clinical chemists are generally confined to measuring the total magnesium concentration in serum (tMg_s)¹ However, around 1990, ion-selective electrodes for determining ionized Mg in blood became available (1). In our laboratory we evaluated one of these electrodes and subsequently established reference values with it (2)(3). This method measures the biologically active Mg fraction, which possibly gives more reliable information on the functional Mg status. However, because serum Mg represents <1% of total body Mg, several techniques for measuring intracellular Mg in many kinds of tissues and cells have been published (4)(5)(6).

In 1991, Ng et al. described a method for measuring ionized Mg in human lymphocytes, using a Mg-sensitive fluorescent probe in combination with spectrophotometry (7). We tested several aspects of this so-called multiple-cell detection method (MCDM) and compared the results by this system with the intracellular ionized Mg concentrations measured by flow cytometry, a so-called single-cell detection method (SCDM). We concluded (see Appendix) that Mg concentrations measured by both techniques are comparable, but we prefer MCDM because it is more easily accessible and less expensive, even though flow cytometry has the advantage of detecting single cells.

In the present study we tried to assess which Mg marker in blood might best indicate deviations from the "normal" condition in healthy subjects. Therefore, we measured extracellular Mg and intracellular Mg, both ionized concentrations and total concentrations, in blood of 23 hemodialysis patients. Known for their disturbed Mg homeostasis (5), these patients represent a model for hypermagnesemia in this study. We then compared the intracellular and extracellular Mg fractions in this specific patient population with the commonly used tMg_s value and with Mg values measured in blood from healthy volunteers.

	Materials and Methods

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subjects
Venous blood used for measurements of ionized and total Mg in serum was drawn into plain, nonsiliconized 4.5-mL tubes. Blood for determining total Mg concentrations in mononuclear blood cells (MBC; tMg_MBC) and in erythrocytes (RBC; tMg_RBC) was collected in 10-mL heparin-containing tubes, and blood for determining ionized Mg in MBC (iMg²_MBC) was drawn into 10-mL tubes containing 0.8 g of polystyrene granules. All tubes used were Vacutainer Tubes (Becton Dickinson).

Reference values for iMg²_MBC were obtained by drawing blood from 40 healthy laboratory workers (16 men, 24 women; median age 36 years, range 21–54 years). Reference values for the other Mg markers had already been established during previous Mg studies in our laboratory (3).

The chronic hemodialysis patients (16 men, 7 women; median age 60 years, range 26–85 years) participating in the study were in a stable condition, hydrated to normal on clinical grounds, and had been treated with hemodialysis for a median of 15 months (range 3–188 months). Underlying renal diseases were chronic glomerulonephritis (n = 14), chronic tubular interstitial nephritis (n = 8), and unknown (n = 1). All patients were treated 2 or 3 times a week with a dialysis solution containing Mg at 0.5 mmol/L (Gambro BV); no MgCO₃ was used as a phosphate-binding agent. Blood was obtained just before treatment was started. All procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 1983.

chemicals
Fluorescent probes [Mag-indo-1/acetoxymethyl ester (AM) and Mag-fura-2/AM] were obtained from Molecular Probes Europe. The ionophore 4-Br-A23187 and nigericin were obtained from Sigma Chemical Co., EDTA and ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) from Boehringer Mannheim GmbH. Other chemicals, all of analytical-reagent grade, were purchased from E. Merck BV.

procedures
Measurement of total and ionized Mg in serum.
After clotting (45 min) and centrifugation (10 min, 1500g), serum was separated from the cells and stored in completely filled rubber-sealed air-tight tubes at -20 °C, for no more than 4 weeks. After thawing, the pH was only slightly increased, so the ionized Mg fraction did not change significantly. tMg_s was measured by atomic absorption spectroscopy (PE2100; Perkin-Elmer), the ionized serum Mg (iMg²_s) by a Mg ion-selective electrode installed in the Microlyte 6 ion analyzer (KONE Instruments).

Isolation of MBC and loading with fluorescent probe.
MBC and RBC used for the determination of the total and ionized intracellular Mg concentration were isolated from heparinized and defibrinated blood, respectively, as described previously (8). After counting and differentiating the cells, we dissolved in 1.0 mL of H₂O the isolated MBC used for the determination of tMg_MBC; for iMg²_MBC determinations, we dissolved the cells in 2.0 mL of buffer containing (in mmol/L): 94.1 NaCl, 23.8 NaHCO₃, 5.64 Na₂HPO₄, 0.42 Ca(NO₃)₂, 5.36 KCl, 0.41 MgSO₄, 11.1 glucose, and 25 HEPES, pH 7.35, plus 50 mL/L fetal calf serum.

We used two different fluorescent probes, loading the cells with Mag-indo-1/AM or Mag-fura-2/AM at final concentrations of 10.9 and 9.4 µmol/L, respectively, by a modification of the method of Raju et al. (9). The cell suspension also contained Pluronic^R 20% (in dimethyl sulfoxide) and probenecid at final concentrations of 0.3 g/L and 1.0 mmol/L, respectively. The main difference between the two fluorescent probes is their difference in emission and excitation behavior. Mag-indo-1 is a dual-emission probe, whereas Mag-fura-2 is a dual-excitation probe. The advantage of the latter is its greater increasing ratio from minimum to maximum Mg concentration, which theoretically allows better precision of the Mg measurement. So, after comparing flow-cytometric intracellular Mg measurements with multiple-cell detection (see Appendix), we used the MCDM with Mag-fura-2 for further measurements.

Measurement of total intracellular Mg concentration.
The total Mg concentration in MBC and RBC was measured as described previously (8). The number of isolated cells was counted with a Bayer-H3 system, the protein concentration of the MBC lysate was measured photometrically after treatment with Coomassie Brilliant Blue (Microprot; Oxford Labware), and Mg was measured by atomic absorption spectrophotometry, as above. The intracellular Mg concentration of MBC was expressed as µmol/g protein; that of RBC, as fmol/cell.

Measurement of intracellular ionized Mg concentration.
The intracellular ionized Mg fraction was determined by using the equation established by Grynkiewicz et al. [10]:

(1)

with K_d the dissociation constant of the probe/Mg complex, R the excitation or emission ratio at the current intracellular Mg concentration, R_min the excitation or emission ratio at minimal intracellular Mg concentration, R_max the ratio at maximal intracellular Mg concentration, S_f the fluorescence intensity of the highest wavelength at R_min, and S_b the fluorescence intensity of the highest wavelength at R_max. After making the cells permeable for Mg by adding the ionophore 4Br-A23187 (final concentration 7.5 µmol/L) and nigericin (final concentration 10 µmol/L), we determined R_max through addition of EDTA/EGTA (final concentration 1.25 mmol/L each) and determined R_max by stepwise addition of increasing concentrations of MgCl₂ (final concentration ~6.5 mmol/L ionized Mg). Because it is impossible to bind all Mg by EDTA/EGTA and saturate all Mag-indo-1 or Mag-fura-2, values for R_min and R_max were approximated by calculations based on the measured ratios, after which iMg²_MBC was calculated by use of Eq. 1 .

Measurements were performed in a 2.0-mL cell suspension (0.3 x 10 to 0.8 x 10 cells/mL) in a 3.0-mL quartz cuvette at 37 °C with a dual excitation and emission spectrophotometer (SLM-Aminco, Aminco Bowman Series 2 luminescence spectrometer). Excitation was at 382 nm (free Mag-fura-2) and 345 nm (bound Mag-fura-2); emission was monitored at 496 nm. K_d, determined by measuring R after the addition of small amounts of MgCl₂ (as described in the Appendix), was found to be 1.44 mmol/L.

statistics
All statistical analyses were performed with the statistical package Statgraphics (PLUSWARE Products). Mg concentrations in different populations were compared by using the two-sample t-test, or the Mann–Whitney U-test if necessary. A two-tailed probability of P <0.05 was considered significant. Correlations between different Mg markers were investigated by the Kendall rank correlation test.

	Results

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comparison of mag-indo-1 and mag-fura-2
Two different fluorescent probes were used for determining iMg²_MBC in the patient population as well as in the group of healthy volunteers. Before pooling the Mg concentrations obtained with both probes, we compared the different values by the two-sample t-test. In neither population, healthy volunteers or hemodialysis patients, was a difference detected between iMg²_MBC measured by Mag-indo-1 and by Mag-fura-2 (hemodialysis patients P = 0.270, healthy volunteers P = 0.490).

mg in hemodialysis patients and healthy volunteers
Table 1 presents the mean total and ionized Mg concentrations in serum and MBC, and the total Mg concentrations in RBC of healthy volunteers and 23 hemodialysis patients. The ranges (mean ± 2 SD) of the intracellular Mg concentrations were much wider than those for Mg in serum. Nevertheless, all Mg markers, including the intracellular fractions, of the patient population were significantly greater than those of the healthy population. tMg_RBC showed the largest increase, namely, 1.5 times the mean value for the healthy volunteers.

There was a correlation between tMg_s and iMg²_s, but also between the two serum Mg markers and tMg_RBC in the blood of the hemodialysis patients. The Kendall rank correlation coefficient () was 0.517 (P = 0.0003) for tMg_s vs tMg_RBC and 0.352 (P = 0.0138) for iMg²_s vs tMg_RBC. No correlation was detected between iMg²_s and iMg²_MBC or between the other Mg markers.

We divided the hemodialysis patient group into two populations, based on whether the tMg_s concentration was below or above the upper reference limit of 1.0 mmol/L. Table 2 presents the blood Mg values for each group. Treatment, duration, and frequency of dialysis of both populations were comparable. With respect to the serum Mg markers and tMg_RBC, the mean values differed significantly (P0.05) between the two populations, but not so for tMg_MBC and iMg²_MBC.

When comparing hemodialysis group 1 (tMg_s concentration 1.0 mmol/L) with the healthy population, all intracellular Mg measures were significantly greater in the patient group, despite both groups' having the same tMg_s value (Fig. 1 ). The iMg²_s concentration of this patient group differed from normal also (P = 0.038), but not as significantly as the intracellular Mg fractions (for tMg_MBC , P = 0.003; for tMg_RBC , P <0.001; and for iMg²_MBC, P = 0.003). In hemodialysis group 2 (tMg_s >1.0 mmol/L), all Mg markers except tMg_MBC were increased in comparison with the healthy population.

View larger version (21K): [in this window] [in a new window]   Figure 1. Concentrations of tMgs, iMg2+s, iMg2+MBC, and tMgRBC (left ordinate) and tMgMBC (right ordinate) in group 1 of the hemodialysis patients (), compared with those in healthy volunteers (). Horizontal lines indicate the mean values for both the control group and the patient group.

	Discussion

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comparison of mag-indo-1 and mag-fura-2
The measurements performed with Mag-indo-1 and Mag-fura-2 in both the healthy volunteers and the hemodialysis patients lead us to conclude that these fluorescent probes measure comparable intracellular Mg concentrations. This conclusion is supported by preceding studies with two calcium probes, Indo-1 and Fura-2, which also detected no difference (11)(12). For the present study, therefore, we pooled the iMg²_MBC concentrations measured with both probes.

mg in hemodialysis patients and healthy volunteers
The human body contains ~1 mol of Mg, <1% of which is present in blood. It is important to determine, therefore, which of the proposed Mg markers gives the best information about the Mg status of the body. Must clinical chemists continue reporting tMg_s; should iMg²_s become the measurement of choice; or is none of the current measurable serum markers appropriate? If none of these, might intracellular Mg markers give proper information about hyper- and hypomagnesemia (possibly depending on the type of disease)? In attempts to answer this question, several studies about measurement and correlations between Mg in serum, RBC, MBC, and tissues (e.g., skeletal muscle, heart, and bone) have been performed. Several reviews on this subject also have been published (13)(14)(15). Although the collective results are still contradictory, to date tMg_MBC seems to give the most appropriate estimate of intracellular Mg. In this study we focused on measurements in serum and blood cells, and evaluated the value of two new Mg markers (iMg²_s and iMg²_MBC) for establishing whether Mg excess was present. We measured these two Mg markers and the total Mg concentration in serum, MBC, and RBC in blood of chronic hemodialysis patients. Because the kidneys play a major role in Mg homeostasis (16), chronic hemodialysis patients are often hypermagnesemic, the extent depending on the Mg concentration in the dialysis solution applied. Because of their Mg overload, these patients form a suitable model for this study (5)(17).

In comparison with the mean Mg values measured in the healthy population, all Mg markers in patients, including the biologically active fractions, were greater, but not all ratios of the Mg markers between the two groups were equal (Table 1 ). Only the increases in the two serum markers were exactly the same (1.27). This means that in this patient population, iMg²_s , the biologically active fraction, is not kept at a more constant value than tMg_s, and the two fluctuate in the same manner. Moreover, even if an active mechanism such as transmembrane Mg channels in MBC might exist to keep the ionized Mg concentration in the cell within certain limits, it does not work properly in this patient group. The ionized fraction in MBC is even further increased than the total intracellular concentration in this type of blood cells. Remarkably, the mean iMg²_MBC concentration in patient group 2 (tMg_s >1.0 mmol/L) was comparable with that in patient group 1 (tMg_s 1.0 mmol/L) (Table 2 ). Therefore, the increase of iMg²_MBC concentration seems to be restricted. No correlation between the increase of both ionized intracellular and extracellular Mg (by 33% and 27%, respectively) was noticed. This finding is supported by Niemela et al. (18), who measured ionized Mg in platelets of healthy adult volunteers and compared these values with several variables, including tMg_s and iMg²_s.

Mag-fura has been used in two other studies to measure ionized Mg in blood cells of patients with renal insufficiency. Kisters et al. (19) determined cytosolic free Mg in lymphocytes of 12 patients with renal insufficiency and compared these values with those of 15 controls. They demonstrated that tMg_s and iMg²_s concentrations were greater in the patient population, whereas the concentration of ionized Mg in lymphocytes was comparable in both groups. Kaupke et al. (20) measured the intracellular ionized fraction in platelets of 9 patients with end-stage renal disease and an increased tMg_s , who were treated with hemodialysis (dialysate containing 0.5 mmol/L Mg). Surprisingly, the Mg concentration measured in these patients was considerably lower than in the normal group. This finding was explained as probably resulting from various clinical conditions to which patients with end-stage renal disease are particularly prone. The discrepancies between our results and those of Kisters et al. (19) and Kaupke et al. (20) show that the final word on this subject has not yet been spoken.

Because not all dialysis patients studied had increased serum total Mg, we looked at dividing the population into two groups (Table 2 ), according to their tMg_s concentration (cutoff value 1.0 mmol/L). Assuming that all of the patients, including those in group 1 (tMg_s 1.0 mmol/L), had a Mg excess, we could conclude that iMg²_s and the three intracellular Mg markers give more reliable information about the Mg status of the tested patients than does the currently used total serum Mg data (Fig. 1 ). On the other hand, despite the difference between the mean values of iMg²_s , which was significant, the measured concentrations overlapped considerably, the mean iMg²_s concentration in the group 1 hemodialysis patients falling within the 2 SD range of the control (healthy volunteer) population. This finding, including an identical increase of iMg²_s and tMg_s seen in comparing hemodialysis patients with the control population, minimizes the clinical usefulness of this Mg measurement as a marker of a Mg excess.

Of the three intracellular markers, iMg²_MBC and tMg_RBC deviated more from the reference value than did tMg_MBC. Even in patient group 2 (tMg_s >1.0 mmol/L), tMg_MBC did not differ significantly from that seen in the control population, probably as a result of the high CV for this Mg assay (14%) (8). tMg_RBC values in this specific patient population result from several influences. In uremic patients RBC have a decreased life span, which results in increased erythropoiesis. Because young RBC contain much more Mg than older cells, hemodialysis patients can be expected to have an increased tMg_RBC (21)(22)(23). Moreover, the increased serum Mg concentration during erythropoiesis can enhance this effect. In our study, tMg_RBC correlated with extracellular Mg (tMg_s and iMg²_s). On the other hand, an increased rate of Na/Mg antiport in hemodialysis patients, which leads only to Mg efflux out of RBC, partially compensates for the intracellular increase (24). In our opinion, all these influences combine to make RBC Mg an unsuitable measure of Mg overload in hemodialysis patients.

The remaining intracellular Mg marker, iMg²_MBC , is not able to discriminate fully between normal Mg homeostasis and Mg excess (Fig. 1 ). Although this measurement showed the largest increase in the hemodialysis population (Table 1 ), and its mean concentration in patient group 1 was significantly increased (Table 2 ), the mean ± 2 SD concentration range for this patient group enclosed the reference interval for the control population. Therefore, although the two ionized Mg measures, iMg²_s and iMg²_MBC , seem to be better indicators in establishing the Mg overload in hemodialysis patients than is the regular measured tMg_s concentration, the ideal marker has yet to be found. Moreover, because the measurement of iMg²_MBC is rather complicated, this intracellular Mg marker is not really suitable as a routine laboratory test.

We therefore conclude that in discriminating between normal Mg homeostasis and Mg excess, the two ionized Mg markers offer minimal advantage; rather, the total Mg concentration in serum remains the marker of choice in hemodialysis patients.

View larger version (15K): [in this window] [in a new window]   Figure A1. Fig. A1. The logarithmic difference as a function of the logarithmic sum of the intracellular ionized magnesium concentrations determined by both methods (SCDM and MCDM).

	Acknowledgments

 
We thank KONE Instruments for providing us with the Microlyte 6 ion-selective analyzer and its consumables and for technical support.

	Footnotes

 
¹ Nonstandard abbreviations: RBC, erythrocytes; MBC, mononuclear blood cells; tMg_s, total serum magnesium; iMg²⁺_s, ionized serum magnesium; tMg_RBC, total magnesium in RBC; tMg_MBC, total magnesium in MBC; iMg²⁺_MBC, ionized magnesium in MBC; iMg²⁺_L , ionized magnesium in lymphocytes; SCDM, single-cell detection method; MCDM, multiple-cell detection method; FSC, forward scatter; SSC, sideway scatter; AM, acetoxymethyl ester; EGTA, ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid.

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