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Centre de Recherche en Nutrition Humaine d’Auvergne, Unité Maladies Métaboliques et Micronutriments, INRA, Centre de Recherche de Theix, 63122 St Genès Champanelle, France
1To whom correspondence should be addressed. E-mail: feillet{at}clermont.inra.fr.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • magnesium • biomarker • status • stable isotope
Magnesium (Mg) is the fourth most abundant cation in the body, and the most abundant intracellular divalent cation. It is involved in a large variety of biological functions and enzymatic reactions (e.g., kinases, phosphorylases, dehydrogenases) (1 ). Mg deficiency leads to severe biochemical changes, and Mg depletion has been reported in many chronic illnesses including neuromuscular disorders, alcoholism, diabetes mellitus and cardiovascular diseases (1 –3 ). In developed countries, dietary Mg generally does not meet the recommended intake for adults, and this might increase the prevalence of Mg deficiency (4 ,5 ). However, despite the importance of Mg to nutrition and health, there are no adequate, sensitive and specific markers to assess Mg status in humans. Mg status is currently evaluated using various biochemical markers (total and ionized plasma Mg, blood cell Mg, skeletal muscle and bone biopsy Mg). However, they all present some limitations. The parenteral Mg loading test has been also suggested as a dynamic test with which to assess Mg status. Recently, determination of exchangeable Mg pool masses using a Mg stable isotope has been proposed as a new approach to evaluate Mg status (6 –9 ). However, these tests are not easily applicable to large populations (10 ). Previous experiments demonstrated that tissue uptake after intraperitoneal injection with 28Mg discriminated between Mg-deficient rats and control rats. In fact, tissues from the deficient rats had higher concentrations of isotope than those of the controls (11 ,12 ). These experiments prompted us to test the hypothesis that uptake of a Mg stable isotope may be higher in cells from Mg-deficient rats than from controls. Thus, we explored Mg status with an in vitro blood load test using a Mg stable isotope. For this purpose, rats with adequate and deficient Mg status were studied. The proposed in vitro blood test was also performed in healthy volunteers.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Male Wistar rats (IFFA-CREDO, L’Arbresle, France), aged 6 wk and weighing 
180 g, were randomly divided into two groups of 10. They were housed under conditions of constant temperature (20–22°C), humidity (45–50%) and a standard dark cycle (2000–0800 h). Our institutional guidelines for the care and use of laboratory animals were observed.
The rats received either the control diet (control group) or a Mg-deficient diet (Mg-deficient group) for 1 mo. The semipurified diets contained the following (g/kg): casein 200, sucrose 650, maize oil 50, alphacel (cellulose) 50, DL-methionine 3, choline bitartrate 2, modified AIN-76 mineral mix 35, AIN-76A vitamin mix 10 (ICN Biomedicals, Orsay, France) (13 ). Mg was omitted from the Mg-deficient diet and was added as MgO to the Mg-adequate diet to attain 1000 mg Mg/kg. Mg concentrations of the diets were determined by chemical analysis and were 55 and 989 mg/kg for the Mg-deficient and -adequate diets, respectively. Distilled water and food were consumed ad libitum. Blood was taken from pentobarbital-anesthetized rats at the abdominal artery into heparinized tubes. An aliquot of blood was centrifuged at 1,000 x g for 10 min and the plasma was separated for total and free Mg determination. Erythrocytes were washed with saline solution (9 g/L NaCl) and hemolyzed in distilled water for total Mg determination. In parallel, the in vitro stable isotope Mg loading test was performed on fresh blood.
Human study.
Blood was taken by venipuncture and collected into heparinized tube from 10 healthy volunteers (4 men and 6 women; age, 40 ± 10 y; body mass index, 22.1 ± 2.4 kg/m2). Blood samples were taken by a professional, experienced nurse. An aliquot of blood was used for blood Mg assessment. The blood was centrifuged at 1,000 x g for 10 min and the plasma was separated for total Mg determination. Erythrocytes were washed with saline solution and hemolyzed for total Mg determination. Another aliquot of blood was used for performing the in vitro stable isotope Mg loading test.
In vitro 25Mg loading test of blood cells.
Rat or human fresh blood (6.5 mL) was incubated with 25Mg stable isotope (0.42 mmol 25Mg/L blood) at 37°C for 2 h after venipuncture. Erythrocytes, lymphocytes and platelets were then isolated for determining total Mg and 25Mg levels.
Isolation of erythrocytes.
An aliquot of incubated blood (0.5 mL) was centrifuged at 1500 x g (10 min, 20°C). Erythrocytes were then washed twice with saline solution (9 g/L), the buffy coat was removed and erythrocytes were hemolyzed in distilled water (1:10), and stored at -20°C until analysis.
Isolation of platelets and lymphocytes.
Briefly, the incubated blood was centrifuged at 150 x g (15 min, 20°C). Plasma-rich platelets were collected on the top of the tube and then centrifuged at 1000 x g (15 min, 20°C). Lymphocytes were isolated according to the method of English and Andersen (14 ). Erythrocytes were first diluted with Hanks’ solution (Sigma-Aldrich, L’Isle d’Abeau Chesnes, France). A double gradient was formed by layering an equal volume of histopaque-1083 or histopaque-1077 (for rats or humans, respectively) over histopaque-1119 (Sigma-Aldrich); the diluted erythrocytes were carefully layered onto the upper medium and then centrifuged at 700 x g (30 min, 20°C). The layer of lymphocytes was carefully removed at the supernatant/histopaque-1083 or histopaque-1077 interphase.
Counting of cells.
The purity of cell preparations was determined by using an automatic hematology cell counter (Cobas, Hoffmann La Roche, Neuilly-sur-Seine, France). Cell smears were prepared to determine the purity after staining with May-Grünwald reagent (Merck, Darmstadt, Germany). The platelet fraction obtained was contaminated with fewer than 1% leukocytes. The lymphocyte preparation contained 98% lymphocytes and 0.7 ± 0.2 platelets/lymphocyte. No erythrocytes were observed in platelet or lymphocyte preparations.
Analysis.
The 25Mg and 26Mg contents of erythrocytes, lymphocytes and platelets were determined by inductively coupled plasma/mass spectrometry (ICP/MS) (PlasmaQuad II systems, Fisons Instruments, Manchester, UK) (15
). Before analysis, lymphocytes and platelets were sonicated. The cell suspensions of hemolyzed erythrocytes, sonicated lymphocytes and platelets were diluted in 1% HNO3 (Suprapur, Merck), and natural Mg and beryllium were used as external and internal standards, respectively. Total Mg concentrations in the analyzed samples were adjusted to 50 µg/L. Within- and between-run percentage residual SD were 0.45 and 0.71% for 25Mg/26Mg for the Mg standard solution and 0.58 and 0.86% for the plasma dilution, respectively.
The 25Mg enrichment of erythrocytes, lymphocytes and platelets (relative and net enrichments) were then calculated according to the following equations:
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Net 25Mg enrichment was then expressed in mmol/L cells for erythrocytes or in µmol/1012 cells for lymphocytes and platelets. For total Mg determination in plasma and erythrocytes, plasma and hemolyzed erythrocytes were diluted adequately in 0.1% lanthanum chloride. Mg concentration was determined by atomic absorption spectrophotometry (Perkin Elmer 560, St Quentin en Yvelines, France) at 285 nm. Appropriate quality controls (seronorm serum, Nycomed, Oslo, Norway) were assayed with each set of measurements. Within- and between-assay percentage residual SD were 2.5 and 3.71% for Mg standard solution, 3.2 and 5.1% for plasma Mg and 3.7 and 5.6% for erythrocyte hemolysate Mg, respectively. Total Mg concentrations of lymphocytes and platelets were obtained from ICP/MS results. Plasma free Mg was determined using an AVL 988/4 analyzer (AVL Medical Instruments, Eragny, France).
Statistical analysis.
Results are means ± SD. The statistical significance of differences between means was assessed using Student’s t test. The limit of statistical significance was set at P < 0.05. Statistical analyses were performed using the GraphPad program (V3.00, GraphPad Software, San Diego, CA).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The weight of Mg-deficient rats at the end of the experiment was significantly lower than that of controls (266 ± 11 vs. 308 ± 22 g, respectively, P < 0.001).
Plasma total and free Mg concentrations were significantly lower in Mg-deficient rats than in controls (Table 1 ). Moreover, the percentage of plasma free Mg was lower in Mg-deficient rats than in controls (33 vs. 66% respectively).
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Interestingly, the relative and net 25Mg enrichments were significantly higher in erythrocytes, lymphocytes and platelets from Mg-deficient rats compared with control rats. The relative and net 25Mg enrichments were increased 100% in lymphocytes and platelets, and 50% (net) and 200% (relative) in erythrocytes from Mg-deficient rats.
Human study.
Total and ionized plasma Mg levels were within the normal range (Table 2 ) (16 ). Erythrocyte Mg levels also were normal. The human erythrocyte 25Mg enrichment percentage ranged from 1.6 to 5.7% with a mean of 2.8%. This relative enrichment percentage in human erythrocytes was lower than that obtained in rat erythrocytes. In lymphocytes and platelets, 25Mg enrichment ranged from 37.7 to 74.9% and from 31.5 to 71.91%, respectively, and 25Mg enrichment in lymphocytes was correlated with 25Mg enrichment in platelets (r = 0.9210, P < 0.01).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Various biochemical markers of Mg status are available. However, they all have some limitations. Serum Mg represents <1% of total body Mg; thus the serum Mg concentration may not reflect intracellular magnesium status. Because ionized Mg is physiologically active, the measurement of ionized Mg would be useful. Mg-deficient rats had lower plasma total and free Mg levels than controls, and free Mg was 66% of total Mg in control rats, but decreased to 33% in Mg-deficient rats. This suggests that plasma ionized Mg is more sensitive to Mg deficiency than plasma total Mg. However, this result differs from that of Zimmermann et al. (17 ) who observed a significant increase in the percentage of plasma free Mg in Mg-deficient (70 mg Mg/kg diet) Sprague-Dawley rats. Therefore, it is difficult to conclude from these results whether plasma ionized Mg can serve as a Mg status biomarker. Measurement of intracellular Mg should better reflect Mg status. The total Mg content of blood cells has been proposed to be an index of Mg status (18 ). Correlation of the Mg blood constituents was inconsistent (10 ) and may vary under different physiologic and pathologic conditions (19 ). In this study, the erythrocyte Mg level was significantly decreased in Mg-deficient rats, but lymphocyte and platelet Mg levels were not. In accordance with our results, Geven et al. (20 ) did not observe any modification of Mg levels in mononuclear cells of Mg-depleted dogs compared with controls. Moreover, Gueux et al. (21 ) made the same observation concerning polymorphonuclear (PMN) blood cells of Mg-deficient rats. In contrast, Ryan et al. (22 ) reported a slight (9%) decrease in Mg content of lymphocytes in Mg-deficient rats, but the decrease was much smaller than that of serum (71%) and erythrocytes (33%). All of these results suggest that lymphocytes, PMN blood cells and platelets may have protective mechanisms against intracellular Mg deficiency and that the determination of total Mg content in leukocytes and platelets to assess Mg status is of questionable usefulness. Therefore, there is still a need to find an accurate biomarker of Mg status.
With that goal, we proposed an in vitro blood load test using the Mg stable isotope. Previous experiments using radioactive 28Mg demonstrated increased uptake of 28Mg by tissues from Mg-deficient rats compared with control rats after an intraperitoneal injection of 28Mg (11
,12
). However the greater uptake by tissues of Mg-deficient rats may have been due to the higher specific activity of their plasma. To counteract this uncertainty, we developed an in vitro test with the use of the stable isotope. This test is based on the hypothesis that cellular demands for Mg increase during Mg deficiency, thus leading to an increase in in vitro cellular uptake of isotopic Mg. Blood cells were chosen because these are easily obtainable in humans. In accordance with the tested hypothesis, our results demonstrated that blood cells (erythrocytes, lymphocytes, platelets) from Mg-deficient rats had significantly increased relative and net 25Mg enrichments compared with control rats. It is rather surprising that the Mg content of lymphocytes and platelets was not modified by Mg deficiency, whereas 25Mg uptake was significantly increased. Modification of cellular Mg flux during Mg deficiency might explain such results. Only 5–10% of intracellular Mg is free Mg2+ and changes in cellular Mg2+ content produce changes in cellular Mg flux (23
). It is therefore possible that lymphocytes and platelets from Mg-deficient rats had a lower Mg2+ level with no modification of total Mg content, i.e., that there was a redistribution of Mg within the cell. This was observed in migraine patients, i.e., the free lymphocyte Mg level was decreased but total lymphocyte Mg content was not modified in patients compared with controls (24
).
To verify whether the in vitro blood load test is applicable to humans, we performed a preliminary study in healthy adult volunteers. We observed that 25Mg enrichment was low for human erythrocytes compared with rats. This is in agreement with the reported low permeability of human RBC membranes to Mg (25 ). In humans, previous erythrocyte Mg exchange studies were conducted after an artificial increase of intracellular Mg and in nonphysiologic medium (26 ). Our study was performed under more physiologic conditions and confirmed previous observations. On the other hand, high 25Mg enrichments were obtained in lymphocytes and platelets. These results suggest that the examination of Mg status in humans with the proposed approach may be more appropriate with lymphocytes and platelets than erythrocytes.
In conclusion, our results demonstrated that the proposed in vitro blood load test using the Mg stable isotope may represent a practical method for examining whole-body Mg status. In fact, relative and net 25Mg enrichments were significantly increased in erythrocytes, lymphocytes and platelets of Mg-deficient rats compared with control rats, reflecting an increasing cellular demand for Mg in Mg deficiency. The observation that 25Mg enrichment was increased in lymphocytes and platelets from Mg-deficient rats, in spite of the absence of modification of total Mg content, emphasizes the usefulness of the stable isotope technique in detecting Mg deficiency. More studies are required to validate the utilization of this test as a biomarker of Mg status in humans.
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ACKNOWLEDGMENTS |
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Manuscript received 3 October 2002. Initial review completed 14 November 2002. Revision accepted 17 December 2002.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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21. Gueux, E., Bussière, F. & Rayssiguier, Y. (2001) Magnesium content of polymorphonuclear blood cells is not an indicator of magnesium status. Rayssiguier, Y. Mazur, A. Durlach, J. eds. Advances in Magnesium Research: Nutrition and Health 2001:259-261 John Libbey & Co London, UK. .
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24. Thomas, J., Millot, J. M., Sebille, S., Delabroise, A. M., Thomas, E., Manfait, M. & Arnaud, M. J. (2000) Free and total magnesium in lymphocytes of migraine patients—effect of magnesium-rich mineral water intake. Clin. Chim. Acta 295:63-75.[Medline]
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