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] Zinc Affects Calcium and Magnesium Retention 221

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 zinc- deficient 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- 222 Nielsen

Table 1 Composition of Basal Diet

Ingredient g/kg

Egg white powder 200.0 Sucrose 232.0 Corn starch 366.5 Safflower oil 100.0 Cellulose 50.0 Choline bitartrate 2.5 L-Cystine 3.0 Vitamin mix, AIN-93 10.0 Mineral mixa 35.0 Biotin mixb 15.0 Total 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 Zinc Affects Calcium and Magnesium Retention 223 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 samplesanalyzedwithanexpectedconcentrationof0.7–1.5 mg creatinine/dL were found to contain 1.18, 1.16, and 1.12 mg/dL. Urine helical peptide and urine and plasma 8-iso- PGF2α 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 ameanof316μg/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 224 Nielsen 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].Proteininliver 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 CCSproteinbandtotheWestern30kDastandard.

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<0.008). However, the low-zinc diet significantly decreased kidney and tibia zinc and plasma cholesterol concentrations compared to both the ad lib and pair-fed 15-mg/kg diets (Table 2). The copper-deficient diet did not significantly affect some variables that change with a severe copper deficiency. As indicated in Table 2, the 1.5-mg copper/kg diet did not affect weight gain or plasma cholesterol of the rats. In addition, Table 3 shows that the low-copper diet did not significantly affect hematocrit and heart weight/body weight ratio. However, the copper-deficient diet decreased plasma copper and ceruloplasmin and kidney copper concentrations and increased liver CCS density ratios (Table 3). Copper deficiency decreased plasma copper most markedly when food intake was restricted by pair-feeding. Zinc Affects Calcium and Magnesium Retention 225

Table 2 Effect of Dietary Copper and Zinc on Biomarkers of Zinc Status

Diet Weight gaina Plasma zinc Kidney zincb Tibia zincb Plasma cholesterol Copper Zinc mg/kg mg/kg G μg/mL μg/g μg/g mg/dL

1.5 8 202±7*† 1.80±0.07 98±2 137±4 50±3 1.5 15 192±7*† 1.76±0.08 106±1 289±5 65±5 1.5 15PFc 184±5*† 1.62±0.05 104±1 273±3 64±4 6.0 8 185±6*† 1.83±0.10 101±1 125±3 47±3 6.0 15 210±9* 1.67±0.05 113±1 287±5 55±3 6.0 15PFd 177±6† 1.60±0.04 112±2 279±8 62±3 Analysis of variance—p values Copper 0.70 0.63 <0.0001 0.51 0.12 Zinc/PF group 0.02e 0.01e <0.0001f <0.0001f 0.0004g Cu×ZnPF group 0.04 0.66 0.12 0.27 0.52

Values presented are the mean±SEM a Values in the column not followed by the same symbols (*, †) are significantly different (p<0.05) according to Tukey’s contrasts b Dry weight basis c Pair-fed (PF) to the rats fed the 1.5-mg copper and 8-mg zinc/kg diets d Pair-fed (PF) to the rats fed the 6.0-mg copper and 8-mg zinc/kg diets e Rats fed the 15-mg zinc/kg diet pair-fed to rats fed the 8-mg zinc/kg diet significantly lower (p=0.008) than rats fed the 8-mg zinc/kg diet according to Tukey’s contrasts f Rats fed the 8-mg zinc/kg diet significantly lower (p=0.0001) than both pair-fed and ad lib rats fed the 15-mg zinc/kg diet according to Tukey’s contrasts g Rats fed the 8-mg zinc/kg diet significantly lower than pair-fed (p=0.0006) and ad lib (p=0.007) rats fed the 15-mg zinc/kg diet according to Tukey’s contrasts

The low-zinc diet affected the urinary excretion of magnesium, calcium, and phosphorus (Table 4). When compared to the pair-fed rats fed the 15-mg zinc/kg diet, rats fed the 8 mg zinc/kg diet exhibited significantly decreased urinary excretion of both magnesium and calcium and significantly increased urinary excretion of phosphorus. The low-copper diet did not significantly affect the urinary excretion of calcium and magnesium. Copper deficiency appeared to increase the urinary excretion of phosphorus, but the difference only approached significance (p=0.06) (Table 4). In contrast to the urinary excretion results, the low-zinc diet did not affect the plasma concentrations of magnesium, calcium, and phosphorus (Table 4). However, the copper-deficient diet increased plasma calcium and phosphorus concentrations (Table 4). Table 5 shows that the zinc-deficient diet increased the magnesium, calcium, and phosphorus concentrations in the tibia; the low-copper diet did not affect these concentrations. Zinc deficiency, but not copper deficiency, also increased kidney magnesium concentration. Unfortunately, because the major objective was to study the effect of marginal zinc deficiency on magnesium metabolism, kidney calcium and phosphorus concentrations were not determined. However, zinc deficiency increased the urinary excretion of helical peptide, which suggests an increased bone turnover and thus a change in calcium utilization. Table 6 shows that the low-copper diet decreased plasma extracellular superoxide dismutase activity and liver glutathione concentration, increased plasma 8-iso-PGF2α, but did not significantly affect liver cytochrome c oxidase activity. The increase in 226 Nielsen

Table 3 Effect of Dietary Copper and Zinc on Biomarkers of Copper Status

Diet Hematocrit Heart weight/body Plasma Plasma Kidney Liver CCS weight coppera ceruloplasmin copper Copper Zinc mg/kg Mg/kg % Ratio×100 ng/mL U/L μg/g Density ratios

1.5 8 41.9±0.5 0.346±0.005 830±72* 48±8 19.3±1.0 0.623±0.077 1.5 15 42.1±0.4 0.336±0.005 1,012±85* 44±9 18.6±0.8 0.816±0.107 1.5 15PFb 41.3±0.5 0.333±0.008 520±111† 28±10 18.3±1.0 0.822±0.121 6.0 8 42.4±0.4 0.337±0.008 1,003±31* 82±6 37.9±3.9 0.589±0.082 6.0 15 42.7±0.4 0.329±0.010 1,025±45* 97±6 49.5±5.2 0.597±0.082 6.0 15PFc 41.6±0.3 0.337±0.006 961±36* 85±3 41.4±1.9 0.613±0.074 Analysis of variance—p values Copper 0.19 0.48 0.0008 <0.0001 <0.0001 0.05 Zinc/PF group 0.12 0.33 0.001 0.17 0.16 0.41 Cu×ZnPF group 0.94 0.56 0.01 0.28 0.11 0.53

Values presented are the mean±SEM CCS copper chaperone for superoxide dismutase a Values in the column not followed by the same symbols (*, †) are significantly different (p<0.05) according to Tukey’s contrasts b Pair-fed (PF) to the rats fed the 1.5-mg copper and 8-mg zinc/kg diets c Pair-fed (PF) to the rats fed the 6.0-mg copper and 8-mg zinc/k g diets

plasma 8-iso-PGF2α was minimal, and urinary 8-iso-PGF2α concentration was decreased by copper deficiency in rats fed the low-zinc diet. The low-zinc diet did not significantly affect plasma extracellular superoxide dismutase activity and plasma 8-iso-PGF2α and liver glutathione concentrations. However, zinc-deprived compared to zinc-adequate pair- fed rats exhibited significantly increased liver cytochrome c oxidase activity.

Discussion

Main Effects of Zinc

The decrease in kidney and tibia zinc concentrations, in addition to a significant decrease in cholesterol concentration without a marked effect on growth, indicates that the 8-mg zinc/ kg diet induced a marginal or subclinical zinc deficiency. When compared to zinc-adequate rats whose diet intake was restricted by 10–15% by pair-feeding, the marginal zinc- deficient rats exhibited significantly decreased magnesium excretion. This finding plus the finding that marginal zinc deficiency increased magnesium concentrations in the tibia and kidney suggest that one of the earliest responses to marginal zinc deficiency is increased magnesium retention. Because marginal zinc deficiency affected urinary calcium excretion and tibia calcium concentrations similar to its effect on tibia and urine magnesium, impaired calcium metabolism or utilization may also be an early response to marginal zinc deficiency. In addition, the increased calcium, phosphorus, and magnesium concentrations in the tibia although helical peptide, an indicator of bone turnover, was increased suggest that calcium utilization by bone cells in the organic matrix was impaired by zinc deficiency. The findings indicating that a marginal zinc deficiency increases magnesium retention and impaired calcium utilization in rats are consistent with increased magnesium balance Zinc Affects Calcium and Magnesium Retention 227

Table 4 Effect of Dietary Copper and Zinc on Urinary Excretion and Plasma Concentrations of Magnesium, Calcium, and Phosphorus

Diet Urine Plasma

Copper Zinc Magnesium Calcium Phosphorus Magnesium Calcium Phosphorus mg/kg mg/kg mg/mmol Cr mg/mmol Cr mg/mmol Cr μg/mL μg/mL μg/mL

1.5 8 19.4±1.7 3.79±0.51 299±19 13.1±0.3 78±2 83±3 1.5 15 22.1±2.5 3.55±0.33 238±23 13.6±0.4 79±1 91±3 1.5 15PFa 34.6±4.0 6.82±1.39 208±13 13.7±0.4 79±1 90±1 6.0 8 23.9±3.1 3.95±0.41 227±21 13.0±0.3 75±2 82±2 6.0 15 23.7±2.8 3.71±0.26 246±27 12.7±0.3 74±2 82±4 6.0 15PFb 36.6±4.1 5.26±1.25 166±30 13.3±0.3 74±1 85±2 Analysis of variance—p values Copper 0.30 0.54 0.06 0.09 0.0002 0.04 Zinc/PF group <0.0001c 0.008d 0.006e 0.33 0.97 0.30 Cu×ZnPF group 0.89 0.48 0.22 0.45 0.81 0.39

Values presented are the mean±SEM a Pair-fed (PF) to the rats fed the 1.5-mg copper and 8-mg zinc/kg diets b Pair-fed (PF) to the rats fed the 6.0-mg copper and 8-mg zinc/kg diets c Rats fed the 15-mg zinc/kg diet pair-fed to rats fed the 8-mg zinc/kg diet significantly higher than rats fed ad lib the 8-mg (p=0.0002) and 15-mg (p=0.0006) zinc/kg diets according to Tukey’s contrasts d Rats fed the 15-mg zinc/kg diet pair-fed to rats fed the 8-mg zinc/kg diet significantly higher than rats fed ad lib the 8-mg (p=0.03) and 15-mg (p=0.01) zinc/kg diets according to Tukey’s contrasts e Rats fed the 15-mg zinc/kg diet pair-fed to rats fed the 8-mg zinc/kg diet significantly lower than rats fed ad lib the 8-mg (p=0.006) and 15-mg (p=0.05) zinc/kg diets according to Tukey’s contrasts and altered bone turnover indicators exhibited by postmenopausal women fed a low-zinc diet (3 mg zinc/day for 90 days) and compared to when dietary zinc intake was 53 mg/day [1]. The basis for marginal zinc deficiency affecting magnesium and calcium metabolism and/or utilization may be the hypothesized defect in cell membranes [2] that results in abnormal uptakes of these mineral elements. Support for the hypothesis of altered cell membranes in marginal zinc deficiency was increased activity of the membrane enzyme, cytochrome c oxidase, in the liver and decreased concentration of an important membrane lipid, cholesterol, in plasma. A change in cell magnesium, which regulates cellular calcium uptake, resulting in an impairment of calcium second-messenger function may be the reason subclinical zinc deficiency results in impaired immune function [12]. The present experiment did not provide any support for the hypothesis that a marginal zinc deficiency results in an abnormal sulfhydryl redox state in the membrane [2]. The marginal zinc deficiency did not affect indicators of oxidative stress, including liver glutathione concentration. However, the lack of support does not negate the hypothesis because direct evidence provided by a change in the sulfhydryl group concentration in a specific cell membrane was not determined in the present study.

Main Effects of Copper

The decrease in plasma copper only when food intake was restricted by pair-feeding or zinc deficiency, moderate decreases in plasma ceruloplasmin activity and kidney copper concentration, increase in liver CCS, and no change in hematocrit or heart weight/body weight ratio indicate that the 1.5-mg copper/kg diet induced only a marginal or subclinical 228 Nielsen

Table 5 Effect of Dietary Copper and Zinc on Tibia (Dry Weight) Concentrations of Magnesium, Calcium, and Phosphorus, Kidney (Dry Weight) Magnesium, and Urinary Excretion of Helical Peptide

Diet Tibia Kidney Urine

Copper Zinc Magnesium Calcium Phosphorus Magnesium Helical peptide mg/kg mg/kg mg/g mg/g mg/g μg/g μg/mmol Cr

1.5 8 3.69±0.07 233±2 113±1 813±6 307±20 1.5 15 3.42±0.04 226±1 109±1 799±9 221±17 1.5 15PFa 3.58±0.04 223±2 108±1 804±7 233±13 6.0 8 3.71±0.09 233±5 112±2 815±8 380±23 6.0 15 3.50±0.08 228±2 109±1 798±5 208±16 6.0 15PFb 3.50±0.06 221±3 107±1 780±7 239±24 Analysis of variance—p values Copper 0.93 0.97 0.58 0.21 0.17 Zinc/PF group 0.003c 0.002d 0.001e 0.01f <0.0001g Cu×ZnPF group 0.49 0.84 0.94 0.17 0.07

Values presented are the mean±SEM a Pair-fed (PF) to the rats fed the 1.5-mg copper and 8-mg zinc/kg diets b Pair-fed (PF) to the rats fed the 6.0-mg copper and 8-mg zinc/k g diets c Rats fed ad lib the 8-mg zinc/kg diet significantly higher (p=0.002) than rats fed ad lib the 15-mg zinc/kg diet according to Tukey’s contrasts d Rats fed ad lib the 8-mg zinc/kg diet significantly higher (p=0.001) than rats fed the 15-mg zinc/kg diet pair-fed to rats fed the 8 mg zinc/kg diet according to Tukey’s contrasts e Rats fed the 15-mg zinc/kg diet pair-fed to rats fed the 8-mg zinc/kg diet significantly higher than rats fed ad lib the 8-mg (p=0.04) and 15-mg (p=0.0009) zinc/kg diets according to Tukey’s contrasts f Rats fed ad lib the 8-mg zinc/kg diet significantly higher (p=0.01) than rats fed the 15-mg zinc/kg diet pair- fed to rats fed the 8-mg zinc/kg diet according to Tukey’s contrasts g Rats fed the 8-mg zinc/kg diet ad lib significantly higher than rats fed the 15-mg zinc/kg diet ad lib and pair-fed the 8-mg zinc/kg diet (p<0.0001) according to Tukey’s contrasts copper deficiency. The marginal copper deficiency apparently increased oxidative stress in the rats because it increased plasma 8-iso-PGF2α and decreased extracellular superoxide dismutase activity and liver glutathione concentration. Unlike marginal zinc deficiency, marginal copper deficiency apparently did not affect magnesium retention or calcium utilization. Marginal copper deficiency apparently affected phosphorus metabolism because it increased plasma phosphorus concentration and tended to increase urinary (p=0.06) phosphorus excretion, especially when diet intake was restricted by pair-feeding or zinc deficiency. This apparent change in phosphorus metabolism may be related to the ATPase, ATP7A (plays a key role in copper transport from the cytosol into secretory pathways) involvement in the activation of extracellular superoxide dismutase [13].

Interactions among Copper, Zinc, and Diet Restriction

A striking finding in the present study was the lack of an interaction between marginal copper and zinc deficiencies that affected oxidative stress, magnesium, and calcium variables examined. However, diet restriction, resulting from pair-feeding or zinc deficiency, affected some responses to marginal zinc and copper deficiencies. Marginal zinc deficiency, which, when severe, induces oxidative stress [14], did not exacerbate oxidative stress induced by marginal copper deficiency. Instead of exacerbating, Zinc Affects Calcium and Magnesium Retention 229

Table 6 Effect of Dietary Copper and Zinc on Indicators of Oxidative Stress

a a Diet ECSOD Plasma 8-iso-PGF2α Urine 8-iso-PGF2α Liver glutathione Liver CCO

Copper Zinc mg/kg mg/kg U/mL ng/mL ng/μmol Cr mmol/g U/mg protein

1.5 8 83±4*# 11.1±0.4*† 1.40±0.14† 6.46±0.34 0.228±0.009 1.5 15 85±4*†# 12.2±0.6*† 1.93±0.14*† 6.92±0.37 0.231±0.005 1.5 15PFb 75±5# 14.0±1.0* 2.09±0.13* 6.54±0.38 0.210±0.006 6.0 8 101±3‡† 11.0±0.4*† 2.02±0.19*† 8.19±0.24 0.229±0.010 6.0 15 99±3‡†* 10.6±0.5† 1.93±0.19*† 7.69±0.32 0.208±0.010 6.0 15PFc 113±4‡ 10.7±1.3† 1.90±0.16*† 7.10±0.35 0.201±0.010 Analysis of variance—p values Copper <0.0001 0.01 0.26 0.0005 0.17 Zinc/PF group 0.88 0.20 0.19 0.24 0.04d Cu×ZnPF group 0.007 0.12 0.04 0.18 0.42

Values presented are the mean±SEM

ECSOD extracellular superoxide dismutase, 8-iso-PGF2α 8-iso-prostaglandin F2α, CCO cytochrome c oxidase a Values in the column not followed by the same symbols (*, #, †, ‡) are significantly different (p<0.05) according to Tukey’s contrasts b Pair-fed (PF) to the rats fed the 1.5-mg copper and 8-mg zinc/kg diets c Pair-fed (PF) to the rats fed the 6.0-mg copper and 8-mg zinc/k g diets d Rats fed ad lib the 8-mg zinc/kg diet significantly higher (p=0.03) than rats fed the 15-mg zinc/kg diet pair- fed to rats fed the 8-mg zinc/kg diet according to Tukey’s contrasts marginal zinc deficiency may have inhibited the oxidative stress response to marginal copper deficiency based on plasma and urine 8-iso-PGF2α concentrations. Measurement of F2-isoprostanes is considered one of the most reliable approaches for assessing oxidative stress in vivo [15]. Marginal zinc deficiency decreased urinary 8-iso-PGF2α excretion and inhibited the increase in plasma 8-iso-PGF2α in copper-deficient rats. The reason for this apparent decrease in oxidative stress is unclear, but may be related to the increased magnesium retention induced by marginal zinc deficiency. Magnesium has antioxidant properties as indicated by magnesium deficiency increasing the susceptibility of lip- oproteins and tissues to peroxidation [16, 17]. In contrast to marginal zinc deficiency, restricting diet intake by 10–15% through pair-feeding enhanced signs of oxidative stress exhibited by marginal copper-deficient rats. Diet restriction by pair-feeding exacerbated the copper deficiency signs of decreased extracellular superoxide dismutase and plasma 8-iso- PGF2α; this exacerbation may have been the result of diet restriction decreasing plasma copper concentration. The exacerbation of copper deficiency signs in marginal deficiency contrasts with the finding of amelioration of severe copper deficiency signs by a 20–30% food restriction [18]. The different response in the more severe copper-deficient model probably resulted from food restriction increasing tissue copper, which may have been related to a decrease in body weight.

Conclusion

Findings from the present experiment did not validate the hypothesis that oxidative stress induced by marginal copper deficiency would exacerbate signs of zinc deficiency by 230 Nielsen increasing oxidative damage in cell membranes. No variable responding to marginal zinc deficiency was significantly altered by marginal copper deficiency. Diet restriction exacerbated the decreased urinary excretion of magnesium and calcium, but did not affect changes in magnesium and calcium concentrations in tissues induced by marginal zinc deficiency. These effects suggest that although diet restriction may alter calcium and magnesium absorption and thus excretion, the changes did not alter the increased magnesium retention and impaired calcium utilization induced by marginal zinc deficiency. This supports the hypothesis that marginal zinc deficiency induces abnormal cellular uptake of magnesium and calcium because of a defect in membrane function, not through changing magnesium and calcium metabolism. Increased cellular magnesium retention and impaired calcium utilization apparently are indicators of marginal zinc deficiency.

Acknowledgements The author thanks Rhonda Poellot and Dale Christopherson for performing the biochemical determinations, Denice Shafer and staff for the animal care, Jim Lindlauf for the animal diet preparation, Craig Lacher and staff for the mineral element analyses, Sheila Bichler and LuAnn Johnson for the statistical analysis assistance, and Martha Haug for the secretarial assistance.

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