Clin Biochem, Vo|. 25, pp. 2 8 9 - 2 9 2 , 1992 Printed in the USA. All rights reserved.
0009-9120/92 $5.00 + .00 Copyright ¢ 1992 The Canadian Society of Clinical Chemists.
Can the Magnesium Content of Mononuclear Blood Cells Be Altered by Oral Magnesium Supplementation ? NORMAN A. DESBIENS, JAMES J. MARX, JR., RONALD G. HAAS, and RICHARD A. REINHART Marshfield Clinic, Marshfield Medical Research Foundation and St. Joseph's Hospital/Marshfield Clinic Joint Venture Laboratory, Marshfield, Wl 54449, USA A randomized, double-blind, placebo-controlled trial was performed on a rigorously defined group of normal subjects to see if magnesium (Mg) supplementation could affect serum Mg levels or Mg content of mononuclear blood cells. Forty-nine subjects were randomized to either placebo, tablets containing 90% United States recommended daily allowance (USRDA) of Mg, or tablets containing 180% USRDA of Mg. We were unable to demonstrate a statistically significant increase in Mg content of mononuclear blood cells.
KEY WORDS: magnesium, mononuclear cells; magnesium, supplementation; magnesium, seasonal variability; magnesium, serum concentration.
Introduction agnesium (Mg) is the fourth most abundant
M cation in the h u m a n body and the second most a b u n d a n t i n t r a c e l l u l a r cation (after potassium). Magnesium is required as a cofactor in the catalysis or activation of over 300 enzymes in the body, including many that are critical in intermediary metabolism. It is also essential for RNA and DNA structure and mitochondrial integrity, as well as neural and myocardial function (1). Hypomagnesemia has been documented in a variety of h u m a n disorders, including intestinal disorders, alcoholism, diuretic use, diabetes, and arrhythmias as well as in patients admitted to the intensive care unit (2,3). However, intracellular depletion has been less well studied. Almost 80% of total body Mg is found in muscle and bone, with only a relatively small amount from these sites available in states of Mg deficiency (4). Also, extracellular Mg represents only 1% of total body Mg. Dissociation between intracellular and extracellular content has been demonstrated in several clinical conditions (5). Temporary fluctuations m a y also occur. T r a n s i e n t de-
Correspondence: Norman A. Desbiens, M.D., Department of Internal Medicine, Marshfield Clinic, 1000 North Oak Avenue, Marshfield, WI 54449, USA. Manuscript received November 1, 1991; revised March 10, 1992; accepted March 12, 1992. CLINICAL BIOCHEMISTRY, VOLUME 25, AUGUST 1992
creases in serum Mg have been demonstrated in patients with myocardial infarction (6). To investigate the role of Mg as an independent variable in h u m a n disease, a test that correlates with intracellular or total body Mg would be helpful. An isotope, 2SMg, has been used in h u m a n studies, but it is expensive, short-lived, and not widely available (7). The Mg loading test has been advocated for clinical use, but its correlation with intracellular Mg is problematic, its performance is inconvenient, its interpretation is difficult, and its usefulness is invalid in many clinical situations (8). A fast, reliable and relatively inexpensive assay for measuring the Mg content of mononuclear blood cells has been developed by several investigators in the hope that Mg in this metabolically active cell would correlate with exchangeable Mg body pools as well as with total body Mg (9,10). Dietary surveys have suggested that the average American diet may be Mg-deficient. This, coupled with the fact that there are no known body storage sites for Mg, has led to the concern that Mg deficiency may be widespread in countries where foodstuffs are highly processed (11). We decided to approach this concern by studying the effects of Mg supplementation on extracellular and intracellular Mg content in a healthy population with no clinical evidence of dietary deficiency. An increase in the content of mononuclear blood cells would be consistent with the hypothesis of widespread Mg deficiency.
Study design The subjects were patients who, after physical exa m i n a t i o n and m u l t i c h a n n e l l a b o r a t o r y testing, were felt by their physicians at the Marshfield Clinic to be in good health and were willing to sign an institutionally-approved consent form. Tablets were allocated in a randomized, doubleblind fashion to be taken once daily as follows: cont r o i s - - 4 0 mg pyridoxine as pyridoxine hydrochloride; group A - - 3 6 2 mg as Mg oxide (90% Adult United States Recommended Daily Allowance 289
DESBIENS, MARX, HAAS, AND REINHART
[USRDA]) (12) and 40 mg pyridoxine as pyridoxine hydrochloride; group B - - 7 2 4 mg as Mg oxide (180% Adult USRDA) and 40 mg pyridoxine as pyridoxine hydrochloride (Mg with pyridoxine tablets [Beelith ®] and placebo tablets supplied by Beach Pharmaceutical, Tampa, FL). After one month, subjects returned to the Clinic for repeat venous blood sampling. Serum Mg was determined by atomic absorption ( P e r k i n - E l m e r Model 306) following a 1:60 dilution with 1% lanthanum chloride solution. Intracellular Mg was determined as described previously (9). This involves cell separation (within 4 h of blood drawing) with Histopaque (Sigma Chemical Co.) and subsequent digestion of the isolated cell pellet with nitric acid. Cell digests were then assayed using conditions as in the serum assay following equi-volume dilution with lanthanum chloride and comparison with matrix-matched aqueous Mg standards. Cell separation for intracellular Mg using heparinized whole blood was performed within 4 h of blood drawing. Day-to-day assay imprecision for serum is 2.5% (1.00 mmol/L). Imprecision on frozen cell suspensions individually digested and assayed on multiple days was 3.6% at a level of 2.96 fmol/cell (9). Pre- and posttreatment results were analyzed using paired t-tests, analysis of variance, and crosstabulation techniques. Two-tailed tests were used throughout. Power calculations indicated a >95% and >75% probability of demonstrating true serum and cellular Mg content differences of 1 standard deviation (SD), respectively.
nificantly different, with p-values ranging from >0.05 to >0.55. Several subgroups analyses were u n d e r t a k e n . Subjects in groups A and B who initially had low mononuclear cell Mg content ( LJ __J
2.0 "-.... • .•.,
....
.'
1.0 X
X
+
JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC 6
10
1
3
12
9
31
45
14
4
0
5
e--
MONTH
F i g u r e 1 - - Splines of mean intracellular Mg level (fmol/cell) and upper and lower 95% confidence limits by month
sampled. Values for March and November are interpolations based on their respective preceding and succeeding months. Symbols above month names indicate significant differences based on overall ~ = 0.05: X months have higher mean Mg levels than * months, and + month has higher mean Mg level than * months and # months (no symbol implies the m e a n Mg level for that month is not significantly different from the other months). The number below each month indicates the number of samples for that month• CLINICAL BIOCHEMISTRY, VOLUME 25, AUGUST 1992
291
DESBIENS, MARX, HAAS, AND REINHART content of mononuclear blood cells and total body Mg content or content of Mg in critical organs, such as the h e a r t or brain, has not yet been demonstrated in humans, although r at studies have suggested a correlation between mononuclear and myocardial Mg content (18).
8.
Acknowledgements
9.
This work was supported in part by Beach Pharmaceuticals, Tampa, FL 33611. The authors thank Kurt Olson, MS, for his assistance with the statistical analyses, Stu Guenther, RPh, for his assistance with drug allocation, Mrs. Nancy Wilke for her assistance with data and subject flow, and Alice Stargardt for manuscript preparation.
10. 11. 12.
References 1. Wacker WEC. Magnesium and man. Cambridge: Harvard University Press, 1980. 2. Reinhart RA. Magnesium metabolism. Arch Intern Med 1988; 148: 2415-20. 3. Reinhart R, Desbiens N. Hypomagnesemia in patients admitted to the intensive care unit. Crit Care Med 1985; 13: 506-7. 4. Dunn MJ, Walser M. Magnesium depletion in normal man. Metabolism 1966; 15: 884-95. 5. Dyckner T, Wester PO. The relation between extraand intracellular electrolytes in patients with hypokalemia and/or diuretic treatment. Acta Med Scand 1987; 204: 269-82. 6. Rasmussen HS, Aurup P, Hojberg S, Jensen EK, McNair P. Magnesium and acute myocardial infarction. Arch Intern Med 1986; 146: 872-4. 7. Wallach S. Magnesium exchangeability and bioavailability in magnesium deficiency. In: Altura BM, Durlach J, Seelig MS, eds. Magnesium and cellular
292
13. 14. 15. 16.
17.
18.
processes in medicine. 4th International Symposium on Magnesium. Pp. 27-49. Blacksburg, VA: S Karger AG, 1985. Caddell JL. The parenteral magnesium retention test. In: Altura BM, Durlach J, Seelig MS, eds. Magnesium and cellular processes in medicine. 4th International Symposium on Magnesium. Pp. 77-88. Blacksburg, VA: S Karger AG, 1985. Reinhart RA, Marx Jr JJ, Haas RG, Desbiens NA. Intracellular magnesium content of mononuclear cells from venous blood of healthy subjects. Clin Chim Acta 1987; 167: 187-95. Elin RJ, Hosseini JM. Magnesium content of mononuclear blood cells. Clin Chem 1985; 31: 377-80. Marier JR. Magnesium content of the food supply in the modern-day world. Magnesium 1986; 5: 1-8. National Research Council. Recommended dietary allowances. 9th ed. Washington: National Academy of Sciences-National Research Council, 1980. Remington's Pharmaceutical Sciences. P. 796. Easton, PA: Mack Publishing Company, 1985. Aikawa JK. Effects of pyridoxine and desoxypyridoxine on magnesium metabolism in the rabbit. Proc Soc Exp Biol Med 1960; 104: 461-3. Kubena KS, Edgar SE, Veltmann JR. Growth and development in rats and deficiency of magnesium and pyridoxine. J A m Coll Nutr 1988; 7: 317-24. Gershoff SN, Prien EL. Effect of daily MgO and vitamin B6 administration to patients with recurring calcium oxalate kidney stones. A m J Clin N u t r 1967; 20: 393-9. Hines JD. Metabolic abnormalities of vitamin B6 and Mg in alcohol-induced sideroblastic anemia. In: Grewer G, ed. Erythrocyte: structure and function. Pp. 621-43. New York: A.R. Liss, 1975. Ryan MF, Ryan MP. Lymphocyte electrolyte alterations during magnesium deficiency in the rat. Ir J Med Sci 1979; 148: 108-9.
CLINICAL BIOCHEMISTRY,VOLUME 25, AUGUST 1992
0009-9120/92 $5.00 + .00 Copyright ¢ 1992 The Canadian Society of Clinical Chemists.
Can the Magnesium Content of Mononuclear Blood Cells Be Altered by Oral Magnesium Supplementation ? NORMAN A. DESBIENS, JAMES J. MARX, JR., RONALD G. HAAS, and RICHARD A. REINHART Marshfield Clinic, Marshfield Medical Research Foundation and St. Joseph's Hospital/Marshfield Clinic Joint Venture Laboratory, Marshfield, Wl 54449, USA A randomized, double-blind, placebo-controlled trial was performed on a rigorously defined group of normal subjects to see if magnesium (Mg) supplementation could affect serum Mg levels or Mg content of mononuclear blood cells. Forty-nine subjects were randomized to either placebo, tablets containing 90% United States recommended daily allowance (USRDA) of Mg, or tablets containing 180% USRDA of Mg. We were unable to demonstrate a statistically significant increase in Mg content of mononuclear blood cells.
KEY WORDS: magnesium, mononuclear cells; magnesium, supplementation; magnesium, seasonal variability; magnesium, serum concentration.
Introduction agnesium (Mg) is the fourth most abundant
M cation in the h u m a n body and the second most a b u n d a n t i n t r a c e l l u l a r cation (after potassium). Magnesium is required as a cofactor in the catalysis or activation of over 300 enzymes in the body, including many that are critical in intermediary metabolism. It is also essential for RNA and DNA structure and mitochondrial integrity, as well as neural and myocardial function (1). Hypomagnesemia has been documented in a variety of h u m a n disorders, including intestinal disorders, alcoholism, diuretic use, diabetes, and arrhythmias as well as in patients admitted to the intensive care unit (2,3). However, intracellular depletion has been less well studied. Almost 80% of total body Mg is found in muscle and bone, with only a relatively small amount from these sites available in states of Mg deficiency (4). Also, extracellular Mg represents only 1% of total body Mg. Dissociation between intracellular and extracellular content has been demonstrated in several clinical conditions (5). Temporary fluctuations m a y also occur. T r a n s i e n t de-
Correspondence: Norman A. Desbiens, M.D., Department of Internal Medicine, Marshfield Clinic, 1000 North Oak Avenue, Marshfield, WI 54449, USA. Manuscript received November 1, 1991; revised March 10, 1992; accepted March 12, 1992. CLINICAL BIOCHEMISTRY, VOLUME 25, AUGUST 1992
creases in serum Mg have been demonstrated in patients with myocardial infarction (6). To investigate the role of Mg as an independent variable in h u m a n disease, a test that correlates with intracellular or total body Mg would be helpful. An isotope, 2SMg, has been used in h u m a n studies, but it is expensive, short-lived, and not widely available (7). The Mg loading test has been advocated for clinical use, but its correlation with intracellular Mg is problematic, its performance is inconvenient, its interpretation is difficult, and its usefulness is invalid in many clinical situations (8). A fast, reliable and relatively inexpensive assay for measuring the Mg content of mononuclear blood cells has been developed by several investigators in the hope that Mg in this metabolically active cell would correlate with exchangeable Mg body pools as well as with total body Mg (9,10). Dietary surveys have suggested that the average American diet may be Mg-deficient. This, coupled with the fact that there are no known body storage sites for Mg, has led to the concern that Mg deficiency may be widespread in countries where foodstuffs are highly processed (11). We decided to approach this concern by studying the effects of Mg supplementation on extracellular and intracellular Mg content in a healthy population with no clinical evidence of dietary deficiency. An increase in the content of mononuclear blood cells would be consistent with the hypothesis of widespread Mg deficiency.
Study design The subjects were patients who, after physical exa m i n a t i o n and m u l t i c h a n n e l l a b o r a t o r y testing, were felt by their physicians at the Marshfield Clinic to be in good health and were willing to sign an institutionally-approved consent form. Tablets were allocated in a randomized, doubleblind fashion to be taken once daily as follows: cont r o i s - - 4 0 mg pyridoxine as pyridoxine hydrochloride; group A - - 3 6 2 mg as Mg oxide (90% Adult United States Recommended Daily Allowance 289
DESBIENS, MARX, HAAS, AND REINHART
[USRDA]) (12) and 40 mg pyridoxine as pyridoxine hydrochloride; group B - - 7 2 4 mg as Mg oxide (180% Adult USRDA) and 40 mg pyridoxine as pyridoxine hydrochloride (Mg with pyridoxine tablets [Beelith ®] and placebo tablets supplied by Beach Pharmaceutical, Tampa, FL). After one month, subjects returned to the Clinic for repeat venous blood sampling. Serum Mg was determined by atomic absorption ( P e r k i n - E l m e r Model 306) following a 1:60 dilution with 1% lanthanum chloride solution. Intracellular Mg was determined as described previously (9). This involves cell separation (within 4 h of blood drawing) with Histopaque (Sigma Chemical Co.) and subsequent digestion of the isolated cell pellet with nitric acid. Cell digests were then assayed using conditions as in the serum assay following equi-volume dilution with lanthanum chloride and comparison with matrix-matched aqueous Mg standards. Cell separation for intracellular Mg using heparinized whole blood was performed within 4 h of blood drawing. Day-to-day assay imprecision for serum is 2.5% (1.00 mmol/L). Imprecision on frozen cell suspensions individually digested and assayed on multiple days was 3.6% at a level of 2.96 fmol/cell (9). Pre- and posttreatment results were analyzed using paired t-tests, analysis of variance, and crosstabulation techniques. Two-tailed tests were used throughout. Power calculations indicated a >95% and >75% probability of demonstrating true serum and cellular Mg content differences of 1 standard deviation (SD), respectively.
nificantly different, with p-values ranging from >0.05 to >0.55. Several subgroups analyses were u n d e r t a k e n . Subjects in groups A and B who initially had low mononuclear cell Mg content ( LJ __J
2.0 "-.... • .•.,
....
.'
1.0 X
X
+
JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC 6
10
1
3
12
9
31
45
14
4
0
5
e--
MONTH
F i g u r e 1 - - Splines of mean intracellular Mg level (fmol/cell) and upper and lower 95% confidence limits by month
sampled. Values for March and November are interpolations based on their respective preceding and succeeding months. Symbols above month names indicate significant differences based on overall ~ = 0.05: X months have higher mean Mg levels than * months, and + month has higher mean Mg level than * months and # months (no symbol implies the m e a n Mg level for that month is not significantly different from the other months). The number below each month indicates the number of samples for that month• CLINICAL BIOCHEMISTRY, VOLUME 25, AUGUST 1992
291
DESBIENS, MARX, HAAS, AND REINHART content of mononuclear blood cells and total body Mg content or content of Mg in critical organs, such as the h e a r t or brain, has not yet been demonstrated in humans, although r at studies have suggested a correlation between mononuclear and myocardial Mg content (18).
8.
Acknowledgements
9.
This work was supported in part by Beach Pharmaceuticals, Tampa, FL 33611. The authors thank Kurt Olson, MS, for his assistance with the statistical analyses, Stu Guenther, RPh, for his assistance with drug allocation, Mrs. Nancy Wilke for her assistance with data and subject flow, and Alice Stargardt for manuscript preparation.
10. 11. 12.
References 1. Wacker WEC. Magnesium and man. Cambridge: Harvard University Press, 1980. 2. Reinhart RA. Magnesium metabolism. Arch Intern Med 1988; 148: 2415-20. 3. Reinhart R, Desbiens N. Hypomagnesemia in patients admitted to the intensive care unit. Crit Care Med 1985; 13: 506-7. 4. Dunn MJ, Walser M. Magnesium depletion in normal man. Metabolism 1966; 15: 884-95. 5. Dyckner T, Wester PO. The relation between extraand intracellular electrolytes in patients with hypokalemia and/or diuretic treatment. Acta Med Scand 1987; 204: 269-82. 6. Rasmussen HS, Aurup P, Hojberg S, Jensen EK, McNair P. Magnesium and acute myocardial infarction. Arch Intern Med 1986; 146: 872-4. 7. Wallach S. Magnesium exchangeability and bioavailability in magnesium deficiency. In: Altura BM, Durlach J, Seelig MS, eds. Magnesium and cellular
292
13. 14. 15. 16.
17.
18.
processes in medicine. 4th International Symposium on Magnesium. Pp. 27-49. Blacksburg, VA: S Karger AG, 1985. Caddell JL. The parenteral magnesium retention test. In: Altura BM, Durlach J, Seelig MS, eds. Magnesium and cellular processes in medicine. 4th International Symposium on Magnesium. Pp. 77-88. Blacksburg, VA: S Karger AG, 1985. Reinhart RA, Marx Jr JJ, Haas RG, Desbiens NA. Intracellular magnesium content of mononuclear cells from venous blood of healthy subjects. Clin Chim Acta 1987; 167: 187-95. Elin RJ, Hosseini JM. Magnesium content of mononuclear blood cells. Clin Chem 1985; 31: 377-80. Marier JR. Magnesium content of the food supply in the modern-day world. Magnesium 1986; 5: 1-8. National Research Council. Recommended dietary allowances. 9th ed. Washington: National Academy of Sciences-National Research Council, 1980. Remington's Pharmaceutical Sciences. P. 796. Easton, PA: Mack Publishing Company, 1985. Aikawa JK. Effects of pyridoxine and desoxypyridoxine on magnesium metabolism in the rabbit. Proc Soc Exp Biol Med 1960; 104: 461-3. Kubena KS, Edgar SE, Veltmann JR. Growth and development in rats and deficiency of magnesium and pyridoxine. J A m Coll Nutr 1988; 7: 317-24. Gershoff SN, Prien EL. Effect of daily MgO and vitamin B6 administration to patients with recurring calcium oxalate kidney stones. A m J Clin N u t r 1967; 20: 393-9. Hines JD. Metabolic abnormalities of vitamin B6 and Mg in alcohol-induced sideroblastic anemia. In: Grewer G, ed. Erythrocyte: structure and function. Pp. 621-43. New York: A.R. Liss, 1975. Ryan MF, Ryan MP. Lymphocyte electrolyte alterations during magnesium deficiency in the rat. Ir J Med Sci 1979; 148: 108-9.
CLINICAL BIOCHEMISTRY,VOLUME 25, AUGUST 1992
