Biol Trace Elem Res (2009) 128:220–231 DOI 10.1007/s12011-008-8268-7
Marginal Zinc Deficiency Increases Magnesium Retention and Impairs Calcium Utilization in Rats Forrest H. Nielsen
Received: 15 October 2008 / Accepted: 22 October 2008 / Published online: 11 November 2008 # Humana Press Inc. 2008
Abstract An experiment with rats was conducted to determine whether magnesium retention is increased and calcium utilization is altered by a marginal zinc deficiency and whether increased oxidative stress induced by a marginal copper deficiency exacerbated responses to a marginal zinc deficiency. Weanling rats were assigned to six groups of ten with dietary treatment variables of low zinc (5 mg/kg for 2 weeks and 8 mg/kg for 7 weeks), low copper (1.5 mg/kg), adequate zinc (15 mg/kg), and adequate copper (6 mg/kg). Two groups of rats were fed the adequate-zinc diet with low or adequate copper and pair-fed with corresponding rats fed the low-zinc diet. When compared to the pair-fed rats, marginal zinc deficiency significantly decreased the urinary excretion of magnesium and calcium, increased the concentrations of magnesium and calcium in the tibia, increased the concentration of magnesium in the kidney, and increased the urinary excretion of helical peptide (bone breakdown product). Marginal copper deficiency decreased extracellular superoxide dismutase and glutathione, which suggests increased oxidative stress. None of the variables responding to the marginal zinc deficiency were significantly altered by the marginal copper deficiency. The findings in the present experiment suggest that increased magnesium retention and impaired calcium utilization are indicators of marginal zinc deficiency. Keywords Zinc . Copper . Magnesium . Calcium . Phosphorus . Oxidative stress
Mention of a trademark or proprietary product does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that also might be suitable. The U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area is an equal opportunity/affirmative action employer, and all agency services are available without discrimination. F. H. Nielsen (*) U.S. Department of Agriculture, Agricultural Research Service, Grand Forks Human Nutrition Research Center, 2420 Second Avenue North, Stop 9034, Grand Forks, ND 58202-9034, USA e-mail:
[email protected]
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Introduction In a controlled metabolic unit study of 21 postmenopausal women, a subclinical deficient zinc intake (3 mg/day) compared to an intake of zinc 32% higher than the upper limit (UL) of 40 mg/day (53 mg/day) decreased the excretion of magnesium in feces and urine, which resulted in increased magnesium balance [1]. The reason for this difference has not been determined, but one possibility suggested was that high dietary zinc impairs the metabolism or utilization of magnesium. Another possibility, which was not presented in that report [1], is that the subclinical or marginal zinc deficiency increased magnesium retention. Although the higher calcium balance in women consuming 3 mg/day was not significantly different from that when they consumed 53 mg/day, other findings suggested that the subclinical deficient intake affected calcium utilization. Both urinary N-telopeptides excretion and serum calcitonin were lower when dietary zinc was 3 mg/day instead of 53 mg/day. These changes suggest that less bone breakdown was needed to maintain calcium homeostasis when dietary zinc was marginally deficient. There are findings suggesting that marginal zinc deficiency affects calcium utilization and magnesium retention. O’Dell [2] has hypothesized that loss of cell membrane zinc resulting in a defect in calcium channels is the first biochemical defect in zinc deficiency and that the defect is caused by an abnormal sulfhydryl redox state in a membrane channel protein. This hypothesis was based on several findings including impaired calcium uptake by glutamate-stimulated brain cortical synaptosomes depolarized with potassium from zincdeficient guinea pigs [3] and addition of glutathione to blood from zinc-deficient rats corrected impaired platelet calcium uptake [4]. It is likely that magnesium uptake by the cell would also be affected by changes in cell membrane function. Magnesium blocks the N-methyl-D-aspartate (NMDA) receptor in cell membranes, which results in an increased threshold level of excitatory amino acids, such as glutamate, to activate this receptor and allow calcium to enter the cell. Thus, increased retention of cellular magnesium may be involved in the impaired calcium uptake by excitable (e.g., platelets and neurons) and nonexcitable cells (i.e., fibroblasts) described by O’Dell [2]. Thus, the following experiment was conducted with rats to determine whether a marginal zinc deficiency increased magnesium retention and altered calcium utilization and whether increased oxidative stress was associated with any change in magnesium retention or metabolism. Because copper deficiency increases oxidative stress and because the human experiment found that a marginal copper intake influenced some responses to the marginal zinc deficiency [1], marginal copper deficiency was made an additional treatment variable.
Materials and Methods Study Design Sixty weanling male Sprague–Dawley rats (Charles River/SASCO, Wilmington, MA, USA) weighing 45–55 g were randomly assigned to groups of ten and fed an AIN-93G diet with dried egg white as the protein source and modified to increase oxidative stress (safflower oil instead of soybean oil and sucrose instead of dextrinized starch) (Table 1) for 9 weeks. Analysis of the basal diet found an average of 1.44 mg copper/kg and 8.25 mg zinc/kg (weeks 3–9). Initially, one treatment variable was dietary zinc at 5 mg/kg. However, after 2 weeks of consuming the 5-mg zinc/kg diet, rats exhibited cyclical consumption of feed and poor growth that indicated a severe zinc deficiency. Thus, zinc in the basal low-
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Table 1 Composition of Basal Diet Ingredient
g/kg
Egg white powder Sucrose Corn starch Safflower oil Cellulose Choline bitartrate L-Cystine Vitamin mix, AIN-93 Mineral mixa Biotin mixb Total
200.0 232.0 366.5 100.0 50.0 2.5 3.0 10.0 35.0 15.0 1,000.0
Analyzed average concentration in the diet of copper was 1.44 mg/kg and of zinc was about 8.25 mg/kg a Composition of the mineral mix (in grams): CaHPO4, 376.4; CaCO3, 83.56; K3(C6H5O7)·H2O, 108.09; MgO, 24.0; Fe(C6H5O7)·5H2O, 6.06; NaSiO2·9H2O, 1.45; MnCO3, 0.63; CuCO3·Cu(OH)2, 0.065; ZnCO3, 0.395; CrK(S04)2·12H2O, 0.275; H3BO3, 0.0815; NaF, 0.0635; 2NiCO3·3Ni(OH)2·4H2O, 0.0318; LiCl, 0.0174; KIO3, 0.0100; (NH4)2MoO4, 0.0080; NH4VO3, 0.0066; and sucrose, 398.8562 b
Composition of the biotin mix (in milligrams): biotin, 1.8; corn starch, 998.2
zinc diet was increased to 8 mg/kg. Dietary variables for the remaining 7 weeks of the experiment were the basal diet containing (per kilogram) 1.5 mg copper and 8 mg zinc and basal diet supplemented (per kilogram) with 4.5 mg copper, 7 mg zinc, or 4.5 mg copper plus 7 mg zinc. Two additional groups of ten rats were fed the diets containing 15 mg zinc and 1.5 mg or 6 mg copper/kg and pair-fed with corresponding rats fed the 8-mg zinc/kg diet. Seven weeks after experiment initiation, each rat was placed in a metabolic cage with free access to drinking water, but not to diet, for a 16-h collection of urine in a plastic tube kept on ice. After 9 weeks, the rats were anesthetized with ether for the collection of blood from the vena cava with a heparin-coated syringe and needle. After euthanasia by decapitation, the right tibia with flesh removed, heart, kidney, and liver were removed. Urine, plasma (obtained by centrifugation), tibias, kidneys, and livers were stored at −70°C until analysis. Animal Handling The rats were housed individually in stainless steel cages in a room maintained at 23°C and 50% humidity with a normal 12-h light and dark cycle. Food was provided in plastic food cups and deionized water (Super Q, Millipore, Bedford, MA, USA) in plastic water bottles with metal tubes. Absorbent paper under the wire mesh cages was changed daily. Rats were weighed and provided clean cages weekly. The Animal Care Committee of the Grand Forks Human Nutrition Research Center approved the study, and lawfully acquired animals were maintained in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals. Analytical Procedures Calcium, magnesium, and phosphorus in undiluted urine as collected were determined by using inductively coupled argon plasma emission spectroscopy (ICAPES) (Optima 3100 XL, Perkin-Elmer, Shelton, CT, USA) that employed a Gem Cone nebulizer with a cyclonic
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spray chamber and an alumina injector tube. Calcium was measured by using line 317.933 nm with a limit of quantification of 0.580 μg/mL. Magnesium was measured by using line 279.077 nm with a limit of quantification of 0.611 μg/mL. Phosphorus was measured by using line 214.914 nm with a limit of quantification of 0.659 μg/mL. Seronorm normal urine (SERO, Billingstad, Norway) was used as the quality control standard; analyzed values obtained were 122±14 μg/mL versus a certified value of 108±4 μg/mL for calcium, 58.3±6.7 μg/mL versus a certified value of 54±3 μg/mL for magnesium, and 665±27 μg/ mL versus a certified value of 590±40 μg/mL for phosphorus. Protein was precipitated from 0.5 mL of plasma by mixing with 0.5 mL of 3.0 N HCl and 1.5 mL of 10% trichloroacetic acid. After allowing the samples to sit for at least 4 h, they were centrifuged at 3,000 rpm for 15 min. The supernatant was analyzed for calcium, copper, magnesium, phosphorus, and zinc by using ICAPES (Optima 3300 DV, Perkin-Elmer, Shelton, CT, USA). UTAK normal range serum (UTAK Laboratories, Valencia, CA, USA) was used as the quality control standard. Analyzed values for calcium, copper, magnesium, and zinc, respectively, were 79±5, 0.83±0.04, 17.4±0.4, and 1.15±0.02 μg/mL versus certified values of 81.5±20.5, 0.72±0.23, 19.0±4.8, and 1.33±0.10 μg/mL for UTAK serum. Diets, tibias (cleaned to the periosteal surface with cheesecloth), and kidneys were lyophilized and then subjected to a wet-ash, low-temperature digestion in Teflon tubes [5]. Calcium, copper, magnesium, phosphorus, and zinc were determined by ICAPES (Optima 3300 DV, Perkin-Elmer, Shelton, CT, USA). Standard reference material (National Institute of Standards and Technology, Gaithersburg, MD, USA) #1577b (bovine liver) was used as the quality control standard. Analyzed values for calcium, copper, magnesium, phosphorus, and zinc, respectively, were 124±12, 166±1.5, 613±64, 11,412±94, and 131±2 μg/g versus certified values of 116±4, 160±8, 600±15, 11,050±350, and 127±16 μg/g for bovine liver. Hematocrit was determined by using a hematology analyzer (Cell-Dyn 3500, Abbott, Chicago, IL, USA). Commercially available kits were used to determine urine creatinine (Creatinine Reagent #83069, Raichem, San Diego, CA, USA), plasma cholesterol (kit #80015, Raichem, San Diego, CA, USA), urine helical peptide (kit #8022, Quidel, San Diego, CA, USA), and urine and plasma 8-iso-prostaglandin F2α(8-iso-PGF2α) (kit 900-091, Assay Designs, Ann Arbor, MI, USA). Urine creatinine analysis was based on the reaction of creatinine with alkaline picrate that forms a color whose absorbance was measured at a wavelength of 510 nm. With this test, control samples analyzed with an expected concentration of 0.7–1.5 mg creatinine/dL were found to contain 1.18, 1.16, and 1.12 mg/dL. Urine helical peptide and urine and plasma 8-isoPGF2α were determined by using competitive immunoassay methods. Helical peptide (or 8-iso-PGF2α) in a sample competes with helical peptide (or 8-iso-PGF2α) conjugated with alkaline phosphatase for a monoclonal antibody. After a reaction with p-nitrophenyl phosphate, the yellow color generated, whose absorbance was measured at 405 nm, was inversely proportional to the concentration of helical peptide in the sample analyzed. Helical peptide determinations of low control samples gave a mean of 55.3 μg/L with a CV of 5.0% versus an expected 42–69 μg/L; determinations of high control samples gave a mean of 316 μg/L with a CV of 3.6% versus an expected 233–380 μg/L. No control sample was supplied with the 8-iso-PGF2α kit. Plasma ceruloplasmin was determined by using the method of Schosinsky et al. [6]. Liver glutathione was determined by using the method of Durand et al. [7]. Extracellular superoxide dismutase activity was determined by assaying the inhibition of acetylated cytochrome c reduction at pH 10.0, as previously described [8, 9]. Liver cytochrome c oxidase activity was measured in liver samples homogenized in ten volumes of buffer containing 0.25 M sucrose, 0.1 mM ethylene
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glycol tetraacetic acid, and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4. Cytochrome c oxidase activity in homogenates was determined by assaying the loss of ferrocytochrome c at 550 nm, as previously described [10]. Protein in liver homogenates was determined by using bicinchoninic acid (BCA Protein Assay Reagent Kit, Pierce, Rockford, IL, USA). Liver copper chaperone for superoxide dismutase (CCS) was determined by using a Western blot method [11]. Liver samples were homogenized in 0.05 M K2HPO4 at pH 7.0 and 0.1% triton X-100 and centrifuged at 13,000×g for 10 min. Proteins (40 μg) were separated by 4–12% Bis–Tris polyacrylamide gel electrophoresis (NuPAGE, Invitrogen, Carlsbad, CA, USA) and then transferred to nitrocellulose membranes (Invitrogen, Carlsbad, CA, USA). Membranes were incubated with rabbit antihuman CCS (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted to 1:300. Membranes were blocked and detected by using Western Breeze detection and secondary antibody kit (WB7105, Invitrogen, Carlsbad, CA, USA). The proteins were scanned and the band intensities were determined by using the Biochem system software (UVP bioimaging system). The CCS bands were standardized by using Magic Mark Western protein standards (Invitrogen, Carlsbad, CA, USA), specifically, the ratios of the CCS protein band to the Western 30 kDa standard. Statistical Analysis Data were statistically analyzed by using 2×3 analysis of variance (SAS/STAT, version 9.1.3, SAS Institute, Cary, NC, USA) with dietary copper and zinc (8, 15, and 15 mg pair-fed) as class variables. Tukey’s contrasts were used to compare group means when appropriate. Values more than two standard deviations from the mean were considered outliers and not included in the analyses. A p value of ≤0.05 was considered statistically significant.
Results The zinc-deficient diet did not significantly affect some variables that change with severe zinc deficiency. The zinc-deficient rats consumed slightly less diet; average daily consumptions for zinc-deficient, pair-fed, and zinc-adequate rats, respectively, were 16.2, 15.2, and 17.3 for rats fed the 1.5-mg copper/kg diet and 14.7, 14.3, and 17.3 for rats fed the 6.0-mg copper/kg diet. However, feeding the 8-mg zinc/kg diet did not significantly affect weight gain of rats over the last 7 weeks of the experiment. The only significant difference determined by a Tukey’s contrast among groups was between ad lib and pair-fed rats fed the 15-mg zinc/kg diet (Table 2). In addition, the low-zinc diet did not decrease plasma zinc (Table 2). Contrarily, the rats fed ad lib the low-zinc diet exhibited significantly higher plasma zinc than the zinc-adequate pair-fed rats (p
