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Effect of hypomagnesemia and exercise on slowly exchanging pools of magnesium
Effect of Hypomagnesemia and Exercise on Slowly Exchanging Pools of Magnesium Lorraine R. Brilla, James H. Fredrickson,
and V. Patteson
Lombardi
The effects of hypomagnesemia and exercise on the slowly exchanging pools of magnesium, skeletal muscle, bone, erythrocytes, and plasma, were examined in four groups of male Sprague-Dawley rats: sedentary-normal diet (SN), exercise-normal diet (EN), sedentary-hypomagnesemic diet @HI. and exercise-hypomagnesemic diet (EH). The exercise groups swam 5 d/wk-’ for 6 weeks. The hypomagnesemic diet contained 80 ppm and the normal diet about 646 ppm of magnesium. Compared with normal-diet rats (SN and EN), dietary-deficient rats (SH and EH) gained less weight and had lower concentrations of magnesium in all tissue samples and plasma (P < .Ol 1. Exercise groups (EN and EH) demonstrated significantly higher magnesium levels in skeletal muscle (P < .Ol 1and a tendency for lower levels of magnesium in plasma, erythrocytes, and bone compared with sedentary groups (SN & SH). EH rats normalized skeletal muscle magnesium, mEq kg-’ wet tissue, (19.9 +_ 5.1) compared with the SN group (20.6 * 2.1). Assuming that magnesium stores that are rapidly exchanged are maintained at the expense of those that are slowly exchanged, magnesium stores in skeletal muscle appear to be most protected, with the effect accentuated by exercise. @ 1989 by Grune & Stratton, inc.
I
NDIVIDUALS in industrialized countries may have inadequate dietary intakes of the nutrient magnesium.‘-4 In the dietary Nationwide Food Consumption Survey conducted in the United States, it was reported that all age/sex classes except for the group 0 to 5 years old were consistently under the recommended daily allowance (RDA) for magnesium, and that 40% of the population had intakes ~70% of the RDA.5.6 Low magnesium intake has been associated with disease processes.‘-” Hypomagnesemia also may be related to physical activity and may be expressed as an inferior performance.‘*,” Numerous studies have been done on animals fed a magnesium-deficient diet. A deficiency of magnesium is known to disrupt carbohydrate,14 lipid,‘S,‘6 and protein metabolism.” Magnesium deficiency also interferes with oxidative phosphorylation by diminishing the production of ATP.15 Studies investigating magnesium metabolism under normal conditions have focused on differentiating between slowly exchanging and rapidly exchanging pools of magnesium.” When dietary intakes are adequate, magnesium levels slowly exchange between bone, skeletal muscle, and erythrocytes. The rapidly exchanging pools of magnesium are contained primarily in the heart, liver, intestine, connective tissue, and skin. In a magnesium-deficient state, the rapidly exchanging pools maintain magnesium levels at the expense of the slowly exchanging pools. For example, in a hypomagnesemic state, the heart, a component of the primary survival core, appears to capture magnesium from the slowly exchanging bone store. This is supported by the observation that rats fed a diet moderately deficient in magnesium had significantly reduced concentrations of magnesium in bone.20 It is apparent that magnesium is needed in the complex cell, tissue, organ and system adaptations that occur in response to exercise. This study examined the effects of chronic exercise and dietary magnesium deficiency on slowly exchanging magnesium pools: skeletal muscle, bone, and erythrocytes in the rat model. MATERIALS AND METHODS
Thirty-two male Sprague-Dawley rats (Bantin and Kingman, Fremont, CA), 42 days old, were divided randomly into four groups: Metabolism, Vol38, No 8 (August), 1989: pp 797-800
sedentary-normal diet (SN), exercise-normal diet (EN), sedentaryhypomagnesemic diet (SH), and exercise-hypomagnesemic diet (EH). The animals were housed in pairs in an environmentally maintained room, with a 1ight:dark cycle of 12: 12 and a temperature of 24“C k 1°C. Food and water were given ad libitum, and rats were handled daily to minimize stress. Diet was controlled by giving standard rat chow (Mg 640 ppm) or a purified hypomagnesemic rat chow at 20% of the rat RDA (Mg 80 ppm) obtained from Purina (Chicago). Exercise training was performed over a 6-week period. The rats were swum in a 50-gal stainless steel tank. Fresh water was used for each session and maintained at a temperature between 32°C and 34OC. The program consisted of a 60-minute period of activity for five consecutive days per week for the 6-week program. Neither resistance nor powdered detergents were used to decrease buoyancy. Powdered detergents are relatively ineffective, while resistance may only increase the oxygen consumptions 29% over nonweighted conditions.” The rats were exercised in groups, with the first group finishing its one hour exercise period completely before the second group began exercising. Within each group, rats were placed into the swim tank in pairs at five-minute intervals until the entire group was swimming simultaneously. Sequentially, the same pairs of rats were then removed from the swim tank at 5-minute intervals following their l-hour swim. Thus, within-group rats exercised together as a group for 40 minutes. All rats were weighed twice a week. At the conclusion of the 6-week exercise program and at least 18 hours after the last exercise session, the animals were sedated with an intramuscular (IM) injection containing a mixture of ketaset (ketamine) and rompun (xylazine). One hundred milligrams of Ketaset (Parke-Davis, Morris Plains, NJ) and 20 mg of Rompun (Haver-Lockhart, Shawnee, KS) were administered per kg of body weight. Once sedated, the animals were placed in a supine position, and a IO-mL syringe with a l-in, 20-gauge needle was used for cardiac puncture. To prevent blood coagulation, each syringe was coated with heparin before the cardiac puncture. Animals died due to hypovolemia following withdrawal of 5 to IO mL of mixed venous blood. The needle from the syringe was removed to prevent hemolysis during the transfer of the whole blood to a heparin-coated tube.
From the Applied Physiology Laboratory, University of Oregon, Eugene. Address reprint requests to Lorraine R. Brilla. PhD. 26 Carver, Western Washington University, Bellingham, WA 98225. o 1989 by Grune & Stratton, Inc. 0026-0495/89/3808/0017/$03.00/0 797
798
BRILLA, FREDRICKSON, AND LOMBARDI Table 1. Repeated Measures Analysis of Variance for Body Weight Source of Variation Exercise Diet Exercise by diet Time Exercise by time Diet by time Exercise by diet by time
F-Ratio
Significance
0.15
0.71
102.54
0.01
0.04
0.85
928.88 1.72
assay. Magnesium
concentration
in bone and muscle were expressed
as:
0.01
l
l
0.05
54.53
0.01.
0.87
0.79
*Statistically significant F value.
Blood was then drawn via capillary action for determination of hematocrit in triplicate. Half of the blood was removed and centrifuged to remove the plasma. Both tubes were then frozen at -20°C until the magnesium assay was performed. Each animal’s right femur and gastrocnemius were excised via dissection. The femur was scraped clean of excess tissue and the periosteum was removed. The gastrocnemius was separated from the soleus at the Achilles’ tendon and cleaned of fascia and connective tissue. Samples were then rinsed with distilled water, dried, frozen in liquid nitrogen, packaged in aluminum foil, and stored on dry ice. Magnesium concentration in erythrocytes was determined by subtracting the plasma magnesium concentration from that of hemolyzed whole blood. The resultant value was expressed relative to hematocrit. The tissue samples were weighed. The bone sample was placed in a porcelain crucible and ashed in a muffle furnace at 535°C 2 5“C for 24 hours. The ash was then ground, weighed, and placed in nitric acid for digestion. Likewise, after weighing, the muscle samples were each placed in a beaker containing 2.0 mL of 15.9 mol/L nitric acid. This mixture was covered with parafilm to prevent contamination and was incubated overnight to allow for digestion. The following day, the sample was neutralized with 4.0 mL of 7.95 mol/L potassium hydroxide, then transferred to a volumetric flask. The original beaker was rinsed five times with 0.2 mL deionized water, and the rinsate was added to the volumetric flask, which was then brought to its IO-mL capacity with deionized water. A O.Ol-mL sample was subsequently drawn from the flask for the magnesium
(Mg:‘)
(0.01 L) (s)-1 = M&,
concentration, Mgr+’ = final where Mg:’ = initial magnesium magnesium concentration expressed per tissue sample weight, and s = skeletal muscle or bone sample mass. All magnesium assays were performed calorimetrically following the technique established by Gindler and Heth.** Samples were read in triplicate with a Beckman DU spectrophotometer (Beckman, Fullerton, CA) at 520 nm wavelength. Statistical procedures included a two way ANOVA repeated measures designed to determine differences in weight, with time as the repeated factor. Magnesium values were analyzed by two way ANOVAs for each of the pools. Due to the small sample size used in this study, the statistical package Condescriptives (SPSSX, or Statistical Packages for the Social Sciences, Chicago), was used to examine the data for skewness and kurtosis. Normality was not violated, so parametrical statistical procedures were deemed appropriate. The statistical level of significance was set at P 5 .05.
RESULTS
The comparisons of weight between the rats based on diet, activity, and time are given in Table 1. Significant differences in weight were due to diet, time, and diet by time. These differences are graphically displayed in Fig 1. Increases in weight with time alone would be expected in young rats. Magnesium values are displayed in Table 2, and statistical analysis is presented in Table 3. No significant interaction effects were noted. Significant differences were noted in plasma, erythrocytes, skeletal muscle, and bone due to the different diets. Also, significant differences were demonstrated in greater skeletal muscle magnesium due to the effect of exercise, with a tendency for reductions in the exercise groups in the plasma, erythrocyte, and bone magnesium pools compared with the sedentary rats.
Fig 1. Mean group body weights measured semiweekly (hypomagnesemic diet indicated by dashed line; normal diet indicated by solid line).
MAGNESIUM
POOLS, EXERCISE, AND DIE7
799
Table 2. Magnesium Concentration in Plasma, Erythrocytes, Skeletal Muscle and Bone Plasma (mEq + L-‘1
RBC (mEq . L-‘)(% HCT-’
SkeletalMuscle (mEq . kg-‘)
SN
1.44 & 0.20
20.3 + 3.8
20.6 + 2.1
227.8 * 64.6
EN
1.27 ? 0.13
18.4 + 2.9
22.4
+ 1.9
206.0
+ 75.8
SH
0.82
? 0.14
13.6 + 5.5
14.9 + 3.1
124.3
+ 23.5
EH
0.77
+ 0.08
11.1 + 2.5
19.9 * 5.1
115.6
+ 33.8
DISCUSSION
This study was conducted to examine differences in slowly exchanging pools of magnesium (Fig 2) and total body weights in four groups of rats subjected to two treatments: diet, normal v hypomagnesemic, and activity, sedentary v exercise. Analyses of whole blood, plasma, skeletal muscle, and bone allowed determination of magnesium in the slowly exchanging pools. All slowly exchanging pools of magnesium demonstrated statistically significant reductions of magnesium when comparing diets, 25% RDA v normal dietary magnesium availability. This would be expected and demonstrates that the slowly exchanging pools will lose magnesium. These responses have been noted in other studies using low magnesium intakes.23*24This may imply that the loss could support the rapidly exchanging pools, most importantly those of the brain, heart, and liver, during hypomagnesemia. Further studies are indicated to support this possibility. Other studies indicate that magnesium is reduced in the myocardium in response to magnesium deficiency.25p26 However, the extent to which the slowly exchanging pools of magnesium may have contributed to the amelioration of this response is not well documented. In both normal and hypomagnesemic states, exercise appeared to exert interesting regulatory effects on the magnesium concentrations of the slowly exchanging pools. The exercise groups in both normal and hypomagnesemic diet
Bone ImEq . kg-‘)
groups showed significant shifts of greater magnesium levels in the skeletal muscle tissue. Exercise demonstrates the capacity to aid in the magnesium capturing or protective process of skeletal muscle. It is postulated that the mechanism may be due in part to the effect of catecholamines. Catecholamines are elevated during strenuous effort, and it has been demonstrated that catecholamines facilitate entry of magnesium into soft tissue.” Since catecholamines were not measured in this study, this and other possible mechanisms that enhance entry of magnesium into skeletal muscle in response to exercise remain to be elucidated. In studies where magnesium deficiency but not exercise were manipulated, it has been reported that morphological changes such as disorganization of the sarcoplasmic reticulum, disruption of the z-band, focal fragmentation of the A-band, and apparent loss of the A-I band patterning was lost,28 and impaired uptake of calcium with reduction in intramuscular tension occurs.*’ In contrast to skeletal muscle, all other slowly exchanging pools of magnesium were not significantly altered when comparing the exercise and sedentary groups, although there was a tendency for reduction in those magnesium sources. This study was undertaken with the hypothesis that bone may be the major contributor of magnesium when dietary levels are inadequate when exposed to stress such as exercise. This was not supported by the data; all slowly exchanging pools contributed to the maintenance of the
Table 3. Statistical Analysis Between Groups of Plasma, Erythrocyte, Skeletal Muscle, and Bone Magnesium Concentration Variance Estimate
F-Ratio
Significance
to.001
Plasma Diet
2.43
117.24
Exercise
0.06
2.92
0.096
Interaction
0.05
2.60
0.115
l
Erythrocytes Diet Exercise Interaction
339.58
22.72
34.79
2.33
0.137
0.55
0.04
0.843
to.00
1l
Skeletal muscle Diet
137.38
13.13
0.002*
Exercise
93.59
8.95
0.006”
Interaction
11.74
1.12
0.299
Bone Diet Exercise Interaction
69254.85 57 1.06 1580.19
‘Statistically significant F value.
22.8 1
0.19
0.671
0.52
0.483
l
Fig 2. The schematic diagram illustrates the proposed relationship between slowly and rapidly exchanging pools in normal (solid arrow) and hypomagnesemic (broken arrow) states. Solid boxes indicate more stable pools. whereas dashed boxes denote more mobile pools.
800
BRILLA, FREDRICKSON. AND LOMBARDI
skeletal muscle magnesium pool. Whether the shift of magnesium to the skeletal muscle pool has implications for vital organ magnesium levels has not been reported. Significant differences in weight were demonstrated in diet treatment, but exercise had no significant effect although the exercise groups were consistently of lower weight than the sedentary groups on similar diets. Growth retardation as assessed by total body weight has been reported previously in rats on a hypomagnesemic diet.” The data supported the hypothesis that a hypomagnesemic
diet induces significant losses of magnesium in all slowly exchanging pools. Exercise was a critical factor in determining higher magnesium retention in skeletal muscle regardless of diet, without significantly affecting plasma, erythrocyte, and bone magnesium when compared with rats on similar intakes of magnesium. The results have implications for exercise performance and possibly vital organ magnesium content when low magnesium diet is combined with regular exercise.
REFERENCES 1. Kramer LB, Osis D, Coffey J, et al: Mineral and trace element content of vegetarian diets. J Am Co11 Nutr 3:3-l 1, 1984 2. Marier JR: Magnesium content of the food supply in the modern-day world. Magnesium 5:1-S, 1986 3. McNeil1 DA, Ali PS, Song YS: Mineral analysis of vegetarian, health, and conventional foods: Magnesium, zinc, copper and manganese. Am Diet Assoc 85569-572, 1985 4. Wilkinson SR, Stuedemann JA, Grunes PL, et al: Relation of soil and plant magnesium to nutrition of animals and man. Magnesium 6:74-90, 1987 5. Marier JR: Quantitative factors regarding magnesium status in the modern-day world. Magnesium 1:3-15, 1982 6. Morgan KJ, Stampley GL, Zabik ME, et al: Magnesium and calcium dietary intakes of the U.S. population. J Am Co11 Nutr 4:195-206, 1985 7. Burch GE, Giles TD: The importance of magnesium deficiency in cardiovascular disease. Am Heart J 94:649-657, 1977 8. Dyckner T: Potassium and magnesium depletion in patients with cardiovascular disease. Am J Med 82:l l-17, 1987 9. Ebel H: Role of magnesium in cardiac disease. J Clin Chem Clin Biochem 21:249-265, 1983 10. Rude RK, Singer FR: Magnesium deficiency and excess. Annu Rev Med 32:245-259, 1981 11. Seeling MS: Magnesium Deficiency in the Pathogenesis of Disease. New York, Plenum, 1980 12. Karppanen H: Epidemiological studies on the relationship between magnesium intake and cardiovascular diseases. Artery 9:190-199, 1981 13. Seelig MS, Heggtveit HA: Magnesium interrelationships in ischemic heart disease. A review. Am J Clin Nutr 27:59-79, 1974 14. McNeil1 DA: Effect of magnesium deficiency in the rat on renal glutaminase and three key gluconeogenic enzymes. Nutr Reports Internat 27:1169-l 177, 1983 15. Rayssiguier Y, Gueux E, Weiser D: Effect of magnesium deficiency on lipid metabolism in rats fed a high carbohydrate diet. J Nutr 111:1876-1883, 1981 16. Rayssiguier Y: Magnesium, lipids and vascular diseases. Magnesium 5:182-190, 1986
17. Stryer L: Biochemistry. New York, Freeman, 1981 18. Vitale JJ, Nakamura M, Hegsted DM: The effect of magnesium deficiency on oxidative phosphorylation. J Biol Chem 228:573576,1957 19. Aikawa JK, Gordon GS, Rhoades EL: Magnesium metabolism in human beings: Studies with MG”. J Appl Physiol 15:503507,196O 20. Hurley LS, Crosens G, Theriault LL: Tetratogenic effects of magnesium deficiency in rats. J Nutr 106:1254-1260, 1976 2 1. McCardle WD: Metabolic stress of endurance swimming in the laboratory rat. J Appl Physiol 22:50-54, 1967 22. Gindler EM, Heth DA: Calorimetric determination with bound calmagite of magnesium in human blood serum. Clin Chem 171662-668, 1971 23. Heroux 0, Peter D, Heggtveit HA: Long-term effect of suboptimal dietary magnesium on magnesium and calcium contents of organs, on cold tolerance and on lifespan, and its pathological consequences in rats. J Nutr 107:1640-1652, 1977 24. Fischer PWF, Giroux A, L’Abbe MR, et al: The effects of moderate magnesium deficiency in the rat. Nutr Reprots Internat 24:993-1000, 198 1 25. Fry CH: Measurement and control of intracellular magnesium ion concentration in guinea pig and ferret myocardium. Magnesium 5:306-316, 1985 26. Elwood disease. Artery
PC: Myocardial 9:200-204, 1981
27. Durlach J, Durlach magnesium homeostasis: 1984
magnesium
V: Speculations A hypothesis.
and
ischemic
on hormonal Magnesium
heart
controls of 3: 109- 13 1,
28. Cronin RE, Ferguson ER, Shannon WA, et al: Skeletal muscle injury after magnesium depletion in the dog. Am J Physiol 243:113-120, 1982 29. Sparkar K, Parry DJ, Heggtveit HA: Skeletal myopathy in chronic magnesium depletion. Magnesium Bull 2: 1OS- 113, 198 1 30. Kubena KS, Landmann WA, Carpenter ZL: Suboptimal intake of magnesium in rats. Effects during growth and gestation. Nutr Res 3:385-394, 1983
and V. Patteson
Lombardi
The effects of hypomagnesemia and exercise on the slowly exchanging pools of magnesium, skeletal muscle, bone, erythrocytes, and plasma, were examined in four groups of male Sprague-Dawley rats: sedentary-normal diet (SN), exercise-normal diet (EN), sedentary-hypomagnesemic diet @HI. and exercise-hypomagnesemic diet (EH). The exercise groups swam 5 d/wk-’ for 6 weeks. The hypomagnesemic diet contained 80 ppm and the normal diet about 646 ppm of magnesium. Compared with normal-diet rats (SN and EN), dietary-deficient rats (SH and EH) gained less weight and had lower concentrations of magnesium in all tissue samples and plasma (P < .Ol 1. Exercise groups (EN and EH) demonstrated significantly higher magnesium levels in skeletal muscle (P < .Ol 1and a tendency for lower levels of magnesium in plasma, erythrocytes, and bone compared with sedentary groups (SN & SH). EH rats normalized skeletal muscle magnesium, mEq kg-’ wet tissue, (19.9 +_ 5.1) compared with the SN group (20.6 * 2.1). Assuming that magnesium stores that are rapidly exchanged are maintained at the expense of those that are slowly exchanged, magnesium stores in skeletal muscle appear to be most protected, with the effect accentuated by exercise. @ 1989 by Grune & Stratton, inc.
I
NDIVIDUALS in industrialized countries may have inadequate dietary intakes of the nutrient magnesium.‘-4 In the dietary Nationwide Food Consumption Survey conducted in the United States, it was reported that all age/sex classes except for the group 0 to 5 years old were consistently under the recommended daily allowance (RDA) for magnesium, and that 40% of the population had intakes ~70% of the RDA.5.6 Low magnesium intake has been associated with disease processes.‘-” Hypomagnesemia also may be related to physical activity and may be expressed as an inferior performance.‘*,” Numerous studies have been done on animals fed a magnesium-deficient diet. A deficiency of magnesium is known to disrupt carbohydrate,14 lipid,‘S,‘6 and protein metabolism.” Magnesium deficiency also interferes with oxidative phosphorylation by diminishing the production of ATP.15 Studies investigating magnesium metabolism under normal conditions have focused on differentiating between slowly exchanging and rapidly exchanging pools of magnesium.” When dietary intakes are adequate, magnesium levels slowly exchange between bone, skeletal muscle, and erythrocytes. The rapidly exchanging pools of magnesium are contained primarily in the heart, liver, intestine, connective tissue, and skin. In a magnesium-deficient state, the rapidly exchanging pools maintain magnesium levels at the expense of the slowly exchanging pools. For example, in a hypomagnesemic state, the heart, a component of the primary survival core, appears to capture magnesium from the slowly exchanging bone store. This is supported by the observation that rats fed a diet moderately deficient in magnesium had significantly reduced concentrations of magnesium in bone.20 It is apparent that magnesium is needed in the complex cell, tissue, organ and system adaptations that occur in response to exercise. This study examined the effects of chronic exercise and dietary magnesium deficiency on slowly exchanging magnesium pools: skeletal muscle, bone, and erythrocytes in the rat model. MATERIALS AND METHODS
Thirty-two male Sprague-Dawley rats (Bantin and Kingman, Fremont, CA), 42 days old, were divided randomly into four groups: Metabolism, Vol38, No 8 (August), 1989: pp 797-800
sedentary-normal diet (SN), exercise-normal diet (EN), sedentaryhypomagnesemic diet (SH), and exercise-hypomagnesemic diet (EH). The animals were housed in pairs in an environmentally maintained room, with a 1ight:dark cycle of 12: 12 and a temperature of 24“C k 1°C. Food and water were given ad libitum, and rats were handled daily to minimize stress. Diet was controlled by giving standard rat chow (Mg 640 ppm) or a purified hypomagnesemic rat chow at 20% of the rat RDA (Mg 80 ppm) obtained from Purina (Chicago). Exercise training was performed over a 6-week period. The rats were swum in a 50-gal stainless steel tank. Fresh water was used for each session and maintained at a temperature between 32°C and 34OC. The program consisted of a 60-minute period of activity for five consecutive days per week for the 6-week program. Neither resistance nor powdered detergents were used to decrease buoyancy. Powdered detergents are relatively ineffective, while resistance may only increase the oxygen consumptions 29% over nonweighted conditions.” The rats were exercised in groups, with the first group finishing its one hour exercise period completely before the second group began exercising. Within each group, rats were placed into the swim tank in pairs at five-minute intervals until the entire group was swimming simultaneously. Sequentially, the same pairs of rats were then removed from the swim tank at 5-minute intervals following their l-hour swim. Thus, within-group rats exercised together as a group for 40 minutes. All rats were weighed twice a week. At the conclusion of the 6-week exercise program and at least 18 hours after the last exercise session, the animals were sedated with an intramuscular (IM) injection containing a mixture of ketaset (ketamine) and rompun (xylazine). One hundred milligrams of Ketaset (Parke-Davis, Morris Plains, NJ) and 20 mg of Rompun (Haver-Lockhart, Shawnee, KS) were administered per kg of body weight. Once sedated, the animals were placed in a supine position, and a IO-mL syringe with a l-in, 20-gauge needle was used for cardiac puncture. To prevent blood coagulation, each syringe was coated with heparin before the cardiac puncture. Animals died due to hypovolemia following withdrawal of 5 to IO mL of mixed venous blood. The needle from the syringe was removed to prevent hemolysis during the transfer of the whole blood to a heparin-coated tube.
From the Applied Physiology Laboratory, University of Oregon, Eugene. Address reprint requests to Lorraine R. Brilla. PhD. 26 Carver, Western Washington University, Bellingham, WA 98225. o 1989 by Grune & Stratton, Inc. 0026-0495/89/3808/0017/$03.00/0 797
798
BRILLA, FREDRICKSON, AND LOMBARDI Table 1. Repeated Measures Analysis of Variance for Body Weight Source of Variation Exercise Diet Exercise by diet Time Exercise by time Diet by time Exercise by diet by time
F-Ratio
Significance
0.15
0.71
102.54
0.01
0.04
0.85
928.88 1.72
assay. Magnesium
concentration
in bone and muscle were expressed
as:
0.01
l
l
0.05
54.53
0.01.
0.87
0.79
*Statistically significant F value.
Blood was then drawn via capillary action for determination of hematocrit in triplicate. Half of the blood was removed and centrifuged to remove the plasma. Both tubes were then frozen at -20°C until the magnesium assay was performed. Each animal’s right femur and gastrocnemius were excised via dissection. The femur was scraped clean of excess tissue and the periosteum was removed. The gastrocnemius was separated from the soleus at the Achilles’ tendon and cleaned of fascia and connective tissue. Samples were then rinsed with distilled water, dried, frozen in liquid nitrogen, packaged in aluminum foil, and stored on dry ice. Magnesium concentration in erythrocytes was determined by subtracting the plasma magnesium concentration from that of hemolyzed whole blood. The resultant value was expressed relative to hematocrit. The tissue samples were weighed. The bone sample was placed in a porcelain crucible and ashed in a muffle furnace at 535°C 2 5“C for 24 hours. The ash was then ground, weighed, and placed in nitric acid for digestion. Likewise, after weighing, the muscle samples were each placed in a beaker containing 2.0 mL of 15.9 mol/L nitric acid. This mixture was covered with parafilm to prevent contamination and was incubated overnight to allow for digestion. The following day, the sample was neutralized with 4.0 mL of 7.95 mol/L potassium hydroxide, then transferred to a volumetric flask. The original beaker was rinsed five times with 0.2 mL deionized water, and the rinsate was added to the volumetric flask, which was then brought to its IO-mL capacity with deionized water. A O.Ol-mL sample was subsequently drawn from the flask for the magnesium
(Mg:‘)
(0.01 L) (s)-1 = M&,
concentration, Mgr+’ = final where Mg:’ = initial magnesium magnesium concentration expressed per tissue sample weight, and s = skeletal muscle or bone sample mass. All magnesium assays were performed calorimetrically following the technique established by Gindler and Heth.** Samples were read in triplicate with a Beckman DU spectrophotometer (Beckman, Fullerton, CA) at 520 nm wavelength. Statistical procedures included a two way ANOVA repeated measures designed to determine differences in weight, with time as the repeated factor. Magnesium values were analyzed by two way ANOVAs for each of the pools. Due to the small sample size used in this study, the statistical package Condescriptives (SPSSX, or Statistical Packages for the Social Sciences, Chicago), was used to examine the data for skewness and kurtosis. Normality was not violated, so parametrical statistical procedures were deemed appropriate. The statistical level of significance was set at P 5 .05.
RESULTS
The comparisons of weight between the rats based on diet, activity, and time are given in Table 1. Significant differences in weight were due to diet, time, and diet by time. These differences are graphically displayed in Fig 1. Increases in weight with time alone would be expected in young rats. Magnesium values are displayed in Table 2, and statistical analysis is presented in Table 3. No significant interaction effects were noted. Significant differences were noted in plasma, erythrocytes, skeletal muscle, and bone due to the different diets. Also, significant differences were demonstrated in greater skeletal muscle magnesium due to the effect of exercise, with a tendency for reductions in the exercise groups in the plasma, erythrocyte, and bone magnesium pools compared with the sedentary rats.
Fig 1. Mean group body weights measured semiweekly (hypomagnesemic diet indicated by dashed line; normal diet indicated by solid line).
MAGNESIUM
POOLS, EXERCISE, AND DIE7
799
Table 2. Magnesium Concentration in Plasma, Erythrocytes, Skeletal Muscle and Bone Plasma (mEq + L-‘1
RBC (mEq . L-‘)(% HCT-’
SkeletalMuscle (mEq . kg-‘)
SN
1.44 & 0.20
20.3 + 3.8
20.6 + 2.1
227.8 * 64.6
EN
1.27 ? 0.13
18.4 + 2.9
22.4
+ 1.9
206.0
+ 75.8
SH
0.82
? 0.14
13.6 + 5.5
14.9 + 3.1
124.3
+ 23.5
EH
0.77
+ 0.08
11.1 + 2.5
19.9 * 5.1
115.6
+ 33.8
DISCUSSION
This study was conducted to examine differences in slowly exchanging pools of magnesium (Fig 2) and total body weights in four groups of rats subjected to two treatments: diet, normal v hypomagnesemic, and activity, sedentary v exercise. Analyses of whole blood, plasma, skeletal muscle, and bone allowed determination of magnesium in the slowly exchanging pools. All slowly exchanging pools of magnesium demonstrated statistically significant reductions of magnesium when comparing diets, 25% RDA v normal dietary magnesium availability. This would be expected and demonstrates that the slowly exchanging pools will lose magnesium. These responses have been noted in other studies using low magnesium intakes.23*24This may imply that the loss could support the rapidly exchanging pools, most importantly those of the brain, heart, and liver, during hypomagnesemia. Further studies are indicated to support this possibility. Other studies indicate that magnesium is reduced in the myocardium in response to magnesium deficiency.25p26 However, the extent to which the slowly exchanging pools of magnesium may have contributed to the amelioration of this response is not well documented. In both normal and hypomagnesemic states, exercise appeared to exert interesting regulatory effects on the magnesium concentrations of the slowly exchanging pools. The exercise groups in both normal and hypomagnesemic diet
Bone ImEq . kg-‘)
groups showed significant shifts of greater magnesium levels in the skeletal muscle tissue. Exercise demonstrates the capacity to aid in the magnesium capturing or protective process of skeletal muscle. It is postulated that the mechanism may be due in part to the effect of catecholamines. Catecholamines are elevated during strenuous effort, and it has been demonstrated that catecholamines facilitate entry of magnesium into soft tissue.” Since catecholamines were not measured in this study, this and other possible mechanisms that enhance entry of magnesium into skeletal muscle in response to exercise remain to be elucidated. In studies where magnesium deficiency but not exercise were manipulated, it has been reported that morphological changes such as disorganization of the sarcoplasmic reticulum, disruption of the z-band, focal fragmentation of the A-band, and apparent loss of the A-I band patterning was lost,28 and impaired uptake of calcium with reduction in intramuscular tension occurs.*’ In contrast to skeletal muscle, all other slowly exchanging pools of magnesium were not significantly altered when comparing the exercise and sedentary groups, although there was a tendency for reduction in those magnesium sources. This study was undertaken with the hypothesis that bone may be the major contributor of magnesium when dietary levels are inadequate when exposed to stress such as exercise. This was not supported by the data; all slowly exchanging pools contributed to the maintenance of the
Table 3. Statistical Analysis Between Groups of Plasma, Erythrocyte, Skeletal Muscle, and Bone Magnesium Concentration Variance Estimate
F-Ratio
Significance
to.001
Plasma Diet
2.43
117.24
Exercise
0.06
2.92
0.096
Interaction
0.05
2.60
0.115
l
Erythrocytes Diet Exercise Interaction
339.58
22.72
34.79
2.33
0.137
0.55
0.04
0.843
to.00
1l
Skeletal muscle Diet
137.38
13.13
0.002*
Exercise
93.59
8.95
0.006”
Interaction
11.74
1.12
0.299
Bone Diet Exercise Interaction
69254.85 57 1.06 1580.19
‘Statistically significant F value.
22.8 1
0.19
0.671
0.52
0.483
l
Fig 2. The schematic diagram illustrates the proposed relationship between slowly and rapidly exchanging pools in normal (solid arrow) and hypomagnesemic (broken arrow) states. Solid boxes indicate more stable pools. whereas dashed boxes denote more mobile pools.
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BRILLA, FREDRICKSON. AND LOMBARDI
skeletal muscle magnesium pool. Whether the shift of magnesium to the skeletal muscle pool has implications for vital organ magnesium levels has not been reported. Significant differences in weight were demonstrated in diet treatment, but exercise had no significant effect although the exercise groups were consistently of lower weight than the sedentary groups on similar diets. Growth retardation as assessed by total body weight has been reported previously in rats on a hypomagnesemic diet.” The data supported the hypothesis that a hypomagnesemic
diet induces significant losses of magnesium in all slowly exchanging pools. Exercise was a critical factor in determining higher magnesium retention in skeletal muscle regardless of diet, without significantly affecting plasma, erythrocyte, and bone magnesium when compared with rats on similar intakes of magnesium. The results have implications for exercise performance and possibly vital organ magnesium content when low magnesium diet is combined with regular exercise.
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