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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
Biol Trace Elem Res (2009) 128:220–231
DOI 10.1007/s12011-008-8268-7
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: forrest.nielsen@ars.usda.gov
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-
Zinc Affects Calcium and Magnesium Retention 221
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
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 mix
a
35.0
Biotin mix
b
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): CaHPO
4
, 376.4; CaCO
3
, 83.56; K
3
(C
6
H
5
O
7
)·H
2
O, 108.09;
MgO, 24.0; Fe(C
6
H
5
O
7
)·5H
2
O, 6.06; NaSiO
2
·9H
2
O, 1.45; MnCO
3
, 0.63; CuCO
3
·Cu(OH)
2
, 0.065; ZnCO
3
,
0.395; CrK(S0
4
)
2
·12H
2
O, 0.275; H
3
BO
3
, 0.0815; NaF, 0.0635; 2NiCO
3
·3Ni(OH)
2
·4H
2
O, 0.0318; LiCl,
0.0174; KIO
3
, 0.0100; (NH
4
)
2
MoO
4
, 0.0080; NH
4
VO
3
, 0.0066; and sucrose, 398.8562
b
Composition of the biotin mix (in milligrams): biotin, 1.8; corn starch, 998.2
222 Nielsen
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
F
2α
(8-iso-PGF
2α
) (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-
PGF
2α
were determined by using competitive immunoassay methods. Helical peptide (or
8-iso-PGF
2α
) in a sample competes with helical peptide (or 8-iso-PGF
2α
) 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-PGF
2α
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 creduction at pH 10.0, as previously
described [8,9]. Liver cytochrome coxidase activity was measured in liver samples
homogenized in ten volumes of buffer containing 0.25 M sucrose, 0.1 mM ethylene
Zinc Affects Calcium and Magnesium Retention 223
glycol tetraacetic acid, and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid,
pH 7.4. Cytochrome coxidase activity in homogenates was determined by assaying the
loss of ferrocytochrome cat 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 K
2
HPO
4
at pH 7.0 and 0.1% triton X-100 and centrifuged at 13,000×gfor
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 pvalue 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 3shows 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.
224 Nielsen
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 5shows 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 6shows that the low-copper diet decreased plasma extracellular superoxide
dismutase activity and liver glutathione concentration, increased plasma 8-iso-PGF
2α
,
but did not significantly affect liver cytochrome coxidase activity. The increase in
Table 2 Effect of Dietary Copper and Zinc on Biomarkers of Zinc Status
Diet Weight gain
a
Plasma zinc Kidney zinc
b
Tibia zinc
b
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 15PF
c
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 15PF
d
177± 6†1.60 ± 0.04 112± 2 279± 8 62± 3
Analysis of variance—pvalues
Copper 0.70 0.63 <0.0001 0.51 0.12
Zinc/PF group 0.02
e
0.01
e
<0.0001
f
<0.0001
f
0.0004
g
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
Zinc Affects Calcium and Magnesium Retention 225
plasma 8-iso-PGF
2α
was minimal, and urinary 8-iso-PGF
2α
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-PGF
2α
and
liver glutathione concentrations. However, zinc-deprived compared to zinc-adequate pair-
fed rats exhibited significantly increased liver cytochrome coxidase 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
Table 3 Effect of Dietary Copper and Zinc on Biomarkers of Copper Status
Diet Hematocrit Heart weight/body
weight
Plasma
copper
a
Plasma
ceruloplasmin
Kidney
copper
Liver CCS
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 15PF
b
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 15PF
c
41.6± 0.3 0.337±0.006 961 ±36* 85±3 41.4 ± 1.9 0.613 ±0.074
Analysis of variance—pvalues
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
226 Nielsen
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 coxidase, 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
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 15PF
a
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 15PF
b
36.6± 4.1 5.26± 1.25 166 ± 30 13.3± 0.3 74 ± 1 85± 2
Analysis of variance—pvalues
Copper 0.30 0.54 0.06 0.09 0.0002 0.04
Zinc/PF group <0.0001
c
0.008
d
0.006
e
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
Zinc Affects Calcium and Magnesium Retention 227
copper deficiency. The marginal copper deficiency apparently increased oxidative stress in
the rats because it increased plasma 8-iso-PGF
2α
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,
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 15PF
a
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 15PF
b
3.50± 0.06 221 ± 3 107± 1 780±7 239± 24
Analysis of variance—pvalues
Copper 0.93 0.97 0.58 0.21 0.17
Zinc/PF group 0.003
c
0.002
d
0.001
e
0.01
f
<0.0001
g
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
228 Nielsen
marginal zinc deficiency may have inhibited the oxidative stress response to marginal
copper deficiency based on plasma and urine 8-iso-PGF
2α
concentrations. Measurement of
F
2
-isoprostanes is considered one of the most reliable approaches for assessing oxidative
stress in vivo [15]. Marginal zinc deficiency decreased urinary 8-iso-PGF
2α
excretion and
inhibited the increase in plasma 8-iso-PGF
2α
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-
PGF
2α
; 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
Table 6 Effect of Dietary Copper and Zinc on Indicators of Oxidative Stress
Diet ECSOD Plasma 8-iso-PGF
2α
a
Urine 8-iso-PGF
2α
a
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 15PF
b
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 15PF
c
113 ± 4‡10.7 ± 1.3†1.90 ±0.16*†7.10± 0.35 0.201 ± 0.010
Analysis of variance—pvalues
Copper <0.0001 0.01 0.26 0.0005 0.17
Zinc/PF group 0.88 0.20 0.19 0.24 0.04
d
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-PGF
2α
8-iso-prostaglandin F
2α
,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
Zinc Affects Calcium and Magnesium Retention 229
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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