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Levels and Distribution of Zinc, Copper, Magnesium, and Calcium in Rats Fed Different Levels of Dietary Zinc MOON K. SONG, 1'3'~ NABEEL F. ADHAM,2'4 AND MARVIN E. AMENT3 1Research and 9Vledical Services, Veterans Administration Medical Center Sepulveda, CA, 91343 USA; and 3Departments of Pediatrics and 4h4edicine UCLA School of Medicine Los Angeles, CA 90024 USA
Received July 8, 1986; Accepted August 8, 1986
ABSTRACT Effects of altered dietary zinc on levels of zinc, copper, magnesium, and calcium in organ and peripheral tissues were studied. When rats fed a zinc-deficient diet (1.3 I~g Zn/g) for 28 d were compared with rats fed a control diet (37.5 p,g Zn/g), levels of zinc were slightly lower in plasma, hair, and skin and 50% lower in femur and pancreas, whereas the levels of copper were higher in all tissue except plasma. Magnesium levels were higher than controls in the heart and lower in the spleen, whereas the calcium levels were lower in plasma, lung, spleen, kidney, and skin and strikingly higher in brain, hair, and femur. When rats fed a zinc-supplemented diet (1.0 mg Zn/g) were compared to the same conrols, levels of zinc in these were higher in all organs and peripheral tissues studied, except heart, lung, and liver; copper levels were higher in liver, kidney, and spleen; magnesium levels were significantly higher in the spleen, but were little affected in other tissues, although calcium levels were higher in pancreas, spleen, kidney, and skin and lower in plasma and hair. These data indicate that overall copper organ and peripheral tissue levels are affected inversely, and zinc and calcium levels directly, by zinc nutriture. *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
75
Vol. 1 I, 1986
Song, Adham, and Ament
76
Index Entries: Zinc; copper; calcium; magnesium; rat; zinc-deftciency; zinc-su pplementation.
INTRODUCTION Zinc is a cofactor for protein and RNA synthesis and DNA replication (1,2). It is also an integral part of more than 120 vital metalloenzymes (3,4). Furthermore, abnormalities of zinc homeostasis have been correlated with numerous pathological manifestations, includng acrodermatitis enteropathica (AE) (5, 6), cancer (7,8), hypogonadism (9), anorexia (10,11), and wound healing (12,13). Although the influence of altered zinc nutriture on regulating absorption of zinc, copper, magnesium, and calcium has been studied by many investigators (14-19), the effects of zinc-supplementation and zinc-deficiency on the distribution of these elements in organ and peripheral tissues of rats have not been thoroughly investigated. Since the regulatory mechanisms of zinc homeostasis in animals reside in the gastrointestinal tract and also, potentially, in the cellular membranes, zinc nutriton must be studied not only at the gastrointestinal level but also at the levels of individual organ tissues. In this paper we wish to report on an experiment showing that variation of zinc nutriture influences the concentratons of zinc, copper, magnesium, and calcium in organ and peripheral tissues of rats. The relationships between zinc nutriture and cellular retention of each element by each organ and peripheral tissue are discussed.
MATERIALS AND METHODS Animals Forty-two weanling, female, Sprague-Dawley rats weighing approximately 50 g (Bentine and Kingman Inc., Femont, CA) were divided into three groups of 14 rats and kept in acid washed stainless-steel hangng cages. The first group of rats was fed a zinc-deficient diet containing less than 1.3 p.g Zn/g; the second group, a control diet containing 37.5 la,g Zn/g; and the third group, a zinc-supplemented diet containing 1.0 mg Zn/g. All rats had access to twice-distilled drinking water containing less than 1 i~g Zn/L. According to our analysis, the zinc levels in the zincdeficient diet were 0.727-1.270 I~g Zn/g diet, a value substantially higher than the value (0.5 I,Lg Zn/g diet) the distributor (Taklad Co., Madison, WI) indicated. Zinc levels in the control and zinc-supplemented diets were approximately those the distributor indicated, with small variations and were not expected to contribute to any change of zinc status in the animals. The composition of the test diets is presented in Table 1. To maintain pair-fed experimental conditions by all the groups (zinc-suppleBiological Trace Element Research
VoL 11. 1986
Dietary Zinc and Alineral Distribution
77
TABLE 1 Composition of Zinc-Deficient Test Diet' mg/kg Egg white solids, spray dried Glucose, monohydrate Maize oil Nonnutritive fiber (cellulose) CaHPO/ KCP NaCI' MgS04" MnS04H20" FeS04'7H20 I' KI03"
CuS04" p-Aminobenzoic acid Ascorbic acid, coated (97.5%) Biotin Vitamin Bi2 (0.1% trituration in mannitol) Calcium pantothenate Choline dihydrogen citrate Folic acid Inositol Manadione (vitamin K3) Niacin Pyridoxine hydrochloride Riboflavin Thiamin hydrochloride Dry retinyl palmitate (500,000 U/g) Dry ergocalciferol (500000 U/g) Dry cz-tocopheryl acetate (500 U/g) Maize starch
200,000 634,305.8 100,000 30,000 19,767 2,288.2 778.1 2,475.2 166.2 200 0.4 015.1 110.1 1,016.6 0.4 029.7 066.1 3,496.9 2.0 110.1 049.6 099.1 022.0 022.0 022.0 039.7 4.4 242.3 4,666.9
'in mg/kg. The control diet (37.5 txg Zn/g): 71.0 mg zinc carbonate/kg diet was added to ingredients of the zinc deficient diet; diet with 1 mg Zn/g added: 1917.9 mg zinc cartxmate/kg diet was added to the zinc deficient test diet. 'Analytical reagent. mented, control, and zinc-deficient) of experimental rats, rats were provided only 15 g diet/rat/d for 14 d. However, because of the development of anorexia by rats fed the zinc-deficient diet, the zinc-deficient group c o n s u m e d only 12.6 g diet/rat/d during d 15-18. Therefore, all groups were fed only 10 g diet/rat/d after d 19 until killed on d 28. During the period of feeding, 10 g diet/rat/d, all the experimental groups essentially stopped growing, but did not lose weight. All procedures followed the guidelines for the care and use of laboratory animals established by the Veterans Administration Animal Study Committee. Biological Trace Element Research
Vol. 11. 1986
78
Song, Adham, and Ament
Determination of Zinc, Copper, Magnesium, and Calcium Heparinized blood samples were collected after decapitation by guillotine and immediately centrifuged at 3000g for 10 rain. One part, by volume, of trichloroacetic acid (TCA) solution (950 g/L) was added to nine parts of plasma sample, vortexed, and centrifuged at 3000g for 10 rain. Each of about 0.5-g representative samples of skin, hair, and muscle, and each entire organ (brain, heart, lung, liver, pancreas, kidney, spleen, and femur) were placed in a 12-mL capacity polystyrene test tube. Representative samples of hair were cut with a pair of scissors from right and left dorsal parts of the body. After the removal of the hair, skin samples were also cut with a prewashed pair of scissors from the areas where hair samples had been removed. Muscle tissue samples were collected from both sides of the body after midincision of the abdomen with a pair of scissors. Hair was washed with distilled water before analysis. Femur and hair samples were burned at 900~ C for 4 h in porcelain crucibles placed in a furnace and then suspended in exactly 5-mL 1M hydrochloric acid. Exactly 5.0 mL TCA solution (100 g/L) were added to each tube containing each tissue sample and homogenized in a polytron homogenizer until a uniform suspension of tissue sample was obtained. The homogenized tissue suspension was centrifuged at 3000g for 15 rain. The supernatant fraction was decanted into a 5-mL capacity, acid-washed, borosilicate glass tube. Recovery values and suitability of zinc (20) and copper (21) have been reported previously. Determinations of magnesium and calcium using the TCA precipitation method have been evaluated and validated by Tietz (22). The supernatant represents the zinc concentration in 5 mL TCA solution extracted from each of the tissue samples. The sample (supernatant fraction), containing zinc, copper, magnesium, and calcium, was further diluted with twice-distilled water to meet the range of linearity of absorbance for the specific element to be determined by atomic absorption spectrophotometer. Student's t-tests for the two means were employed for the comparison of the values of zinc, copper, calcium, and magnesium levels in organ and peripheral tissues.
RESULTS The mean body weight of rats fed a zinc-deficient diet was only 67.6% at the time of death and that of rats fed a zinc-supplemented diet, 108.4% of controls (Table 2). The differences from the controls were significant for zinc-deficient rats at the p < 0.001 level and at the p < 0.05 level for zinc-supplemented rats. The relative size of organs as mg/g body weight was greater for all organs studied, except liver, in zinc-deficient rats and in all organs in zinc-supplemented rats, except the femur (Fig. 1). On the other hand, protein concentrations as mg/g tissue decreased in essentially all organ and peripheral tissues of zinc-deficient rats (Fig. 2). Biological Trace Element Research
VoL 1 I, 1986
79
Dietary Zinc and Mineral Distribution
TABLE2 Body-Weights of Rats Fed Control, Zinc-Deficient, and Zinc-Supplemented Diets for 4 wk" Ixg Zn/g diet Zn contents of diets,
0.5, Zn-deficient Mean
Body wt, g % controls Statistical significance of difference
37.5, control
1000, Zn-supplemented
SE
Mean
SE
Mean
69.8 3.8 67.6 3.7 p 0.01
103.3 100.0
3.1 3.0
111.9 3.5 108.4 3.4 p < 0.05
Levels and Distribution of Zinc, Copper, Magnesium, and Calcium in Rats Fed Different Levels of Dietary Zinc MOON K. SONG, 1'3'~ NABEEL F. ADHAM,2'4 AND MARVIN E. AMENT3 1Research and 9Vledical Services, Veterans Administration Medical Center Sepulveda, CA, 91343 USA; and 3Departments of Pediatrics and 4h4edicine UCLA School of Medicine Los Angeles, CA 90024 USA
Received July 8, 1986; Accepted August 8, 1986
ABSTRACT Effects of altered dietary zinc on levels of zinc, copper, magnesium, and calcium in organ and peripheral tissues were studied. When rats fed a zinc-deficient diet (1.3 I~g Zn/g) for 28 d were compared with rats fed a control diet (37.5 p,g Zn/g), levels of zinc were slightly lower in plasma, hair, and skin and 50% lower in femur and pancreas, whereas the levels of copper were higher in all tissue except plasma. Magnesium levels were higher than controls in the heart and lower in the spleen, whereas the calcium levels were lower in plasma, lung, spleen, kidney, and skin and strikingly higher in brain, hair, and femur. When rats fed a zinc-supplemented diet (1.0 mg Zn/g) were compared to the same conrols, levels of zinc in these were higher in all organs and peripheral tissues studied, except heart, lung, and liver; copper levels were higher in liver, kidney, and spleen; magnesium levels were significantly higher in the spleen, but were little affected in other tissues, although calcium levels were higher in pancreas, spleen, kidney, and skin and lower in plasma and hair. These data indicate that overall copper organ and peripheral tissue levels are affected inversely, and zinc and calcium levels directly, by zinc nutriture. *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
75
Vol. 1 I, 1986
Song, Adham, and Ament
76
Index Entries: Zinc; copper; calcium; magnesium; rat; zinc-deftciency; zinc-su pplementation.
INTRODUCTION Zinc is a cofactor for protein and RNA synthesis and DNA replication (1,2). It is also an integral part of more than 120 vital metalloenzymes (3,4). Furthermore, abnormalities of zinc homeostasis have been correlated with numerous pathological manifestations, includng acrodermatitis enteropathica (AE) (5, 6), cancer (7,8), hypogonadism (9), anorexia (10,11), and wound healing (12,13). Although the influence of altered zinc nutriture on regulating absorption of zinc, copper, magnesium, and calcium has been studied by many investigators (14-19), the effects of zinc-supplementation and zinc-deficiency on the distribution of these elements in organ and peripheral tissues of rats have not been thoroughly investigated. Since the regulatory mechanisms of zinc homeostasis in animals reside in the gastrointestinal tract and also, potentially, in the cellular membranes, zinc nutriton must be studied not only at the gastrointestinal level but also at the levels of individual organ tissues. In this paper we wish to report on an experiment showing that variation of zinc nutriture influences the concentratons of zinc, copper, magnesium, and calcium in organ and peripheral tissues of rats. The relationships between zinc nutriture and cellular retention of each element by each organ and peripheral tissue are discussed.
MATERIALS AND METHODS Animals Forty-two weanling, female, Sprague-Dawley rats weighing approximately 50 g (Bentine and Kingman Inc., Femont, CA) were divided into three groups of 14 rats and kept in acid washed stainless-steel hangng cages. The first group of rats was fed a zinc-deficient diet containing less than 1.3 p.g Zn/g; the second group, a control diet containing 37.5 la,g Zn/g; and the third group, a zinc-supplemented diet containing 1.0 mg Zn/g. All rats had access to twice-distilled drinking water containing less than 1 i~g Zn/L. According to our analysis, the zinc levels in the zincdeficient diet were 0.727-1.270 I~g Zn/g diet, a value substantially higher than the value (0.5 I,Lg Zn/g diet) the distributor (Taklad Co., Madison, WI) indicated. Zinc levels in the control and zinc-supplemented diets were approximately those the distributor indicated, with small variations and were not expected to contribute to any change of zinc status in the animals. The composition of the test diets is presented in Table 1. To maintain pair-fed experimental conditions by all the groups (zinc-suppleBiological Trace Element Research
VoL 11. 1986
Dietary Zinc and Alineral Distribution
77
TABLE 1 Composition of Zinc-Deficient Test Diet' mg/kg Egg white solids, spray dried Glucose, monohydrate Maize oil Nonnutritive fiber (cellulose) CaHPO/ KCP NaCI' MgS04" MnS04H20" FeS04'7H20 I' KI03"
CuS04" p-Aminobenzoic acid Ascorbic acid, coated (97.5%) Biotin Vitamin Bi2 (0.1% trituration in mannitol) Calcium pantothenate Choline dihydrogen citrate Folic acid Inositol Manadione (vitamin K3) Niacin Pyridoxine hydrochloride Riboflavin Thiamin hydrochloride Dry retinyl palmitate (500,000 U/g) Dry ergocalciferol (500000 U/g) Dry cz-tocopheryl acetate (500 U/g) Maize starch
200,000 634,305.8 100,000 30,000 19,767 2,288.2 778.1 2,475.2 166.2 200 0.4 015.1 110.1 1,016.6 0.4 029.7 066.1 3,496.9 2.0 110.1 049.6 099.1 022.0 022.0 022.0 039.7 4.4 242.3 4,666.9
'in mg/kg. The control diet (37.5 txg Zn/g): 71.0 mg zinc carbonate/kg diet was added to ingredients of the zinc deficient diet; diet with 1 mg Zn/g added: 1917.9 mg zinc cartxmate/kg diet was added to the zinc deficient test diet. 'Analytical reagent. mented, control, and zinc-deficient) of experimental rats, rats were provided only 15 g diet/rat/d for 14 d. However, because of the development of anorexia by rats fed the zinc-deficient diet, the zinc-deficient group c o n s u m e d only 12.6 g diet/rat/d during d 15-18. Therefore, all groups were fed only 10 g diet/rat/d after d 19 until killed on d 28. During the period of feeding, 10 g diet/rat/d, all the experimental groups essentially stopped growing, but did not lose weight. All procedures followed the guidelines for the care and use of laboratory animals established by the Veterans Administration Animal Study Committee. Biological Trace Element Research
Vol. 11. 1986
78
Song, Adham, and Ament
Determination of Zinc, Copper, Magnesium, and Calcium Heparinized blood samples were collected after decapitation by guillotine and immediately centrifuged at 3000g for 10 rain. One part, by volume, of trichloroacetic acid (TCA) solution (950 g/L) was added to nine parts of plasma sample, vortexed, and centrifuged at 3000g for 10 rain. Each of about 0.5-g representative samples of skin, hair, and muscle, and each entire organ (brain, heart, lung, liver, pancreas, kidney, spleen, and femur) were placed in a 12-mL capacity polystyrene test tube. Representative samples of hair were cut with a pair of scissors from right and left dorsal parts of the body. After the removal of the hair, skin samples were also cut with a prewashed pair of scissors from the areas where hair samples had been removed. Muscle tissue samples were collected from both sides of the body after midincision of the abdomen with a pair of scissors. Hair was washed with distilled water before analysis. Femur and hair samples were burned at 900~ C for 4 h in porcelain crucibles placed in a furnace and then suspended in exactly 5-mL 1M hydrochloric acid. Exactly 5.0 mL TCA solution (100 g/L) were added to each tube containing each tissue sample and homogenized in a polytron homogenizer until a uniform suspension of tissue sample was obtained. The homogenized tissue suspension was centrifuged at 3000g for 15 rain. The supernatant fraction was decanted into a 5-mL capacity, acid-washed, borosilicate glass tube. Recovery values and suitability of zinc (20) and copper (21) have been reported previously. Determinations of magnesium and calcium using the TCA precipitation method have been evaluated and validated by Tietz (22). The supernatant represents the zinc concentration in 5 mL TCA solution extracted from each of the tissue samples. The sample (supernatant fraction), containing zinc, copper, magnesium, and calcium, was further diluted with twice-distilled water to meet the range of linearity of absorbance for the specific element to be determined by atomic absorption spectrophotometer. Student's t-tests for the two means were employed for the comparison of the values of zinc, copper, calcium, and magnesium levels in organ and peripheral tissues.
RESULTS The mean body weight of rats fed a zinc-deficient diet was only 67.6% at the time of death and that of rats fed a zinc-supplemented diet, 108.4% of controls (Table 2). The differences from the controls were significant for zinc-deficient rats at the p < 0.001 level and at the p < 0.05 level for zinc-supplemented rats. The relative size of organs as mg/g body weight was greater for all organs studied, except liver, in zinc-deficient rats and in all organs in zinc-supplemented rats, except the femur (Fig. 1). On the other hand, protein concentrations as mg/g tissue decreased in essentially all organ and peripheral tissues of zinc-deficient rats (Fig. 2). Biological Trace Element Research
VoL 1 I, 1986
79
Dietary Zinc and Mineral Distribution
TABLE2 Body-Weights of Rats Fed Control, Zinc-Deficient, and Zinc-Supplemented Diets for 4 wk" Ixg Zn/g diet Zn contents of diets,
0.5, Zn-deficient Mean
Body wt, g % controls Statistical significance of difference
37.5, control
1000, Zn-supplemented
SE
Mean
SE
Mean
69.8 3.8 67.6 3.7 p 0.01
103.3 100.0
3.1 3.0
111.9 3.5 108.4 3.4 p < 0.05
