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CLIN. CHEM. 23/10, 1834-1837(1977)
Zinc, Iron, Copper, and MagnesiumConcentrationsin Tissuesof
Rats Fed VariousAmountsof Zinc
Han K. Kang, Phillip W. Harvey, Jane L. Valentine, and Marian E. Swendseid
An experiment was conducted with rats to determine the
effects on the tissue concentrations of Zn, Fe, Cu, and Mg
of feeding various amounts of zinc. The rats were pair-fed
one of the following diets for four weeks: Diet A, a zincdeficient diet; a diet containing the recommended amount
of zinc (diet A plus 55 ig of zinc per gram of diet), or diet
A plus 550 tg zinc per gram of diet. Concentrations of
these elements in various tissues were determined by
atomic absorption spectrometry after wet digestion.
Feeding the rats zinc-supplemented diets resulted in increased zinc in blood, heart, kidney, and liver, and a
marked decrease of iron in kidney and liver. Concentrations of the other two elements were unchanged in all
tissues. Thus, the effect of zinc in decreasing iron concentrations in liver, observed by several investigators when
rats were fed toxic amounts of zinc, also occurs when zinc
is administered in normal or subtoxic amounts.
Additional Keyphrases: trace metals
and Fe
.
.
relation between Zn
ferritin
It has long been recognized that there is a partial biological antagonism between zinc and iron and between
zinc and copper (1-3). Smith and Larson (1) found that
feeding young rats excess zinc, from 7000 to 10 000 tg
per gram of diet, caused anemia and subnormal growth.
Three-fourths
of these animals died within three to five
weeks when they were fed 10 000 tg of zinc per gram of
diet. Cox and Harris (2) reported that zinc toxicosis of
rats resulted in an accumulation of zinc in the liver with
an early and marked loss of hepatic iron. Settlemire and
Matrone (3) showed that high zinc intake, 7500 g of
zinc per gram of diet, decreased both the amount of
hepatic ferritin and the percentage of iron bound to
ferritin. The question of whether there is a similar relationship between hepatic zinc and iron content after
feeding much lower amounts of dietary zinc has not
School of Public Health, Center for Health Sciences, U.C.L.A., Los
Angeles, Calif. 90024.
Received May 19, 1977; accepted July 5, 1977.
1834
CLINICAL CHEMISTRY, Vol. 23, No. 10, 1977
been investigated. The nutritional requirement of zinc
for normal growth of weanling rats is reportedly 8 to 65
zg of zinc per gram of diet (4-6).
Our study was initiated to determine the effects of
low, normal, and excess (nontoxic) amounts of dietary
zinc on the content of zinc and other essential elements
in rat tissues.
Materials and Methods
Weanling male Sprague-Dawley
rats weighing 45-60
g were maintained individually in stainless-steel
cages
and given free access to distilled and de-ionized water
in glass bottles. The rats were randomly divided into
three groups. Group A received ad libitum a commercially prepared
(Bio Serv., Inc., Frenchtown,
N. J.
08825) zinc-deficient
diet that contained, by analysis,
1.3 tg of zinc per gram. The diet is described by BioServ. as containing the following (grams per kilogram
of diet): dextrose, 631; cellulose, 30; spray dried egg
white solids, 200; corn oil, 100; salt mix, 37; and vitamin
mix.1 We did not attempt to measure the amount of
phytate in the diet because, according to information
obtained from Bio-Serv., no phytate was present. Group
B received the same zinc-deficient
diet, but supplemented with zinc carbonate equivalent to 55 tg of zinc
per gram, and group C the same diet supplemented with
zinc carbonate equivalent to 550 tg of zinc per gram.
Rats in group B and group C received each day an
amount of diet equal to the diet intake of their pairmates in group A.
After four weeks, the rats were decapitated and blood
1 Salt mix, mg/kg: CaCO3, 9944; CaHPO4, 3149; CoC126H2O, 2;
CuSO4#{149}5H20,
10; ferric citrate, 911; MgSO4.7H20, 3381; MnSO4.H20,
9; KI, 27; K2HPO4, 14 000; NaCL, 5552. Vitamin mix, mg/kg: biotin,
4; B12, 20; calcium pantothenate,
16; choline chloride, 1500; chlortetracycline, 250; folic acid, 0.5; menadione, 0.3; niacin, 25; pyridoxine.HC1, 4; riboflavin, 6; thiaminHCl,
10; vitamin A palmitate, 10 X
106 mt. units; vitamin D2, 1.25 X 10 mt. units; vitamin E, 0.11 X 10
mt. units.
Table 1. Weight Gain and Food Efficiency Ratio of
Rats Fed Zinc-Deficient and Zinc-Supplemented
Diet
Food
Body WI, g
Group
A(8)b
B (7)
C (6)
a
At begInnIng
50.6 ± 5.6c
At end
of
4
ratIo8
76.8 ± 14.9
96.0 ± 16.8
97.0 ± 25.3
50.6 ± 4.6
50.7 ± 5.1
0.15
0.25
0.24
Grams of body weight gain per gram of food consumed.
Figures In parentheses: no. of animals used in each dietary group.
Values are mean ±1 SD.
was sampled from the head wound. Heart, lung, spleen,
liver, and kidney were excised, trimmed of connective
tissue, dried by blotting with filter paper, and weighed.
The tissues were then placed in polyethylene bags and
kept in a freezer until the analysis.
The frozen tissues were placed in an Erlenmeyer flask
for wet ashing. Glass beads, prewashed with nitric acid,
and 20 ml of distilled and de-ionized water were added
to the flask, followed by 10 ml of concentrated nitric acid
and perchloric acid (equal volumes). The samples were
initially heated very gently. After foaming subsided, the
temperature
was increased to produce steady boiling.
If charring occurred, small amounts of nitric acid were
added immediately. The excess acids were boiled off to
near (not complete) dryness. The digests, when cooled
to room temperature,
were diluted to 5 ml with distilled
and de-ionized water, then diluted to bring the concentration within the range of the standard.
Metal concentrations
in the digest were determined
by atomic absorption spectrometry with a Perkin-Ehner
Model 303 atomic absorption
spectrophotometer
equipped with a three-slot burner head, a hollow-cathode lamp, and air and acetylene flame. The following
wavelengths were used: zinc, 213.8; copper, 324.7; iron,
248.3; and magnesium
285.2 nm. Standard
solutions
were purchased from Hartman-Leddon
Co., Philadelphia, Pa. 19143.
For comparison of means between different groups,
Student’s t -test was used.
Dietary
Group
Results
Table 1 summarizes
weight gain and the food efficiency ratio of rats fed various amounts of zinc. The rats
that were receiving diet A (the zinc-deficient diet) were
anorexic by the fifth day of feeding. Rats fed the zincdeficient diet ad libitum had a mean daily diet intake
of 6.8 g per rat. Our preliminary study showed that rats
receiving the diet supplemented
with zinc carbonate
equivalent to 55 tg of zinc per gram consumed a mean
of 12.8 g during the same period. The food efficiency
ratio, defined as grams of body weight gain per gram of
rood consumed, was lowest in the zinc-deficient
rats.
The gross appearance
of the zinc-deficient
rats at the
end of the feeding period was similar to observations of
other investigators
(7-9). Body hair was sparse and
coarse, especially over the abdomen and hind legs. Rats
fed diet B (diet A + 55 g of zinc per gram of diet) and
diet C (diet A + 550 g of zinc per gram of diet) showed
similar weight gains and food efficiency ratios. Table 2
shows the mean organ weights at the time the rats were
killed. Mean hematocrit
and standard
deviation for
groups A, B, and C were 45 ± 2, 49 ± 2, and 47 ± 4, respectively. Table 3 shows the content of Zn, Fe, Cu, and
Mg in various tissues from the rats. The zinc content of
the heart, kidney, liver, and blood was lower in the
zinc-deficient
rats than in the zinc-supplemented
rats.
Copper and magnesium content of all tissues showed
no significant alteration.
The magnesium
content of
blood from zinc-deficient rats was lower than that of the
rats fed 550 g of zinc per gram of diet. Essential-element concentrations
of lung and spleen tissue were the
same in all of the dietary groups, hence they are not
shown in Table 3.
The relationship of zinc and iron content in the liver
was evaluated by the test for independence
method
described by Dixon and Massey (10). The overall correlation coefficient (r) was -.485, significant at the 0.05
level.
Discussion
As mentioned, typical symptoms of zinc deficiency
were observed in the rats of group A. Although the three
Table 2. Organ Weight of Rats Fed Zinc-Deficient and Zinc-Supplemented Diet
Head
Lung
Spleen
Liver
Kidney
Wet WI, 9
A(8)8
B (7)
C (6)
0.36
0.48
0.37
0.38
0.40
0.41
±
±
±
±
±
±
003L
0.08c
0.06
0.03
0.1
0.03
0.57 ±
0.76 ±
0.70 ±
0.75 ±
0.69 ±
0.76 ±
0.03
0.13
0.20
0.25
0.12
0.20
0.18 ±
0.23 ±
0.21 ±
0.22 ±
0.23 ±
0.23 ±
0.04
0.05
0.06
0.04
0.01
0.07
0.92 ±
1.23 ±
0.92 ±
0.97 ±
1.05 ±
1.12 ±
0.04
0.16
0.15
0.14
0.18
0.17
3.00 ±
3.95 ±
3.25 ±
3.30 ±
3.11 ±
3.23 ±
0.39
0.37
0.39
0.25
0.76
0.25
#{149}
Figures In parentheses: no. animals In each dietary group.
Ii Mean organ wet weIght ± SD,grams.
C Mean organ wet weight
relative to body weight ± SD. in percentage.
CLINICALCHEMISTRY,
Vol. 23, No. 10, 1977
1835
Table 3. Conc#{233}#{224}tratlon#{232}
of Zn, Fe, Cu, and Mg in Tissues of Rats Fed Zinc-Deficient and ZincSupplemented Diet
Dietary
group
Cup
on
nc
4119/9 wet
Magnesium
weight a
Heart
A
B
C
12.5 ± 1.1
74.2± 7.5
14.5 ±
14.3±
73.3 ± 7.4
73.1 ± 6.9
17b
13b
4.2 ± 0.4
4.2 ± 0.6
183.8 ± 15.3
178.9 ± 19.4
3.7 ± 0.4
170.4 ± 12.9
Kidney
A
B
C
Liver
A
C
39.7 ±
53b,c
B
18.5 ± 5.7
30.2 ± 61b
C
399 ±
Blood
A
b
23.7 ± 4.3
31.4 ± 73b
71b.c
B
3.2 ± 0.5
4.3 ±
C
5.3 ±
06b.c
52.7 ± 14.7
47.4 ± 66b
6.1 ± 2.4
4.1 ± 0.8
166.4 ± 14.5
187.6 ± 45.6
4.0 ± 0.6
169.7 ± 8.3
339.9± 117
197.7 ± 50.Ob
95.9 ± 25b.c
3.9 ± 1.3
4.5 ± 0.6
4.3 ± 0.9
205.3 ± 27.6
219.6 ± 17.3
60.1 ± 10.2
333.1 ± 53.9
384.3 ± 51.9
380.7 ± 68.1
0.79 ± 0.16
0.77 ± 0.22
0.82 ± 0.12
203.1 ± 41.9
41.1 ± 5.4
44.7 ± 4.1
51.9 ±
94b
Vaiues represent mean ± SD, in ig/g wet weight.
SignifIcantly (P <0.05) different from group A value.
SIgnifIcantly (P <0.05) different from group B value.
groups of rats were pair-fed, the rats fed zinc-supplemented diets showed greater weight gain. Rats in group
B and group C showed similar weight gains and food
efficiency ratios, which suggests that 550 g of zinc per
gram of diet was not toxic for them.
Macapinlac et al. (9) found a significant increase in
hematocrit values in their zinc-deficient
rats. We did
not; our hematocrit values were within the normal range
(11). A previous study by Settlemire and Matrone (3)
suggests that excess dietary zinc may somehow limit the
absorption of iron from the digestive tract. Conversely,
Cox and Harris (2) indicate that excess zinc does not
interfere with iron absorption. If concentrations
of essential elements in the blood reflect absorption from the
digestive tract, we are in agreement with the findings
of Cox and Harris. The values for concentrations
of zinc,
copper, iron, and magnesium in the tissues of rats fed
zinc-supplemented
diets agree with those of control rats
in other studies (2, 3, 5).
The zinc content in liver and kidney increased as the
dietary zinc was increased, but the iron content in these
organs decreased. Toxic levels of dietary zinc have been
reported to result in decreased concentrations
of iron
in liver and kidney (2). Thus, an increase in zinc and a
decrease in iron content evidently occurs not only with
toxic levels of dietary zinc, as reported previously, but
also when zinc is present in the diet in normal or subtoxic levels. Prasad et al. (5) found a similar relationship
between zinc and iron in testes of rats fed zinc-deficient
(10 Mgof zinc per gram of diet) and zinc-supplemented
diets (65 g of zinc per gram of diet). The relatively high
amounts of zinc in their zinc-deficient
diet (10 g zinc
vs. 1.3 pg zinc per gram of diet in the present study) may
explain their failure to observe similar results in other
1838 CLINICALCHEMISTRY,
Vol. 23, No. 10, 1977
tissues. Their study failed to demonstrate
any change
in zinc content in the liver. Addition of phytate (to
contain lOg of phytate per kilogram of diet) to both the
zinc-deficient diet and the control diet in this study may
have also contributed
to the different results. Chen et
al. (12) have reported that the increase in liver zinc
observed after feeding the rats diets supplemented
with
zinc acetate equivalent to either i#{248}00pg
of zinc per gram
of diet or 2000 g of zinc per gram of diet was associated
with increased amounts of zinc appearing in the liver
soluble fraction. This increased zinc content in the
soluble fraction was mainly attributable
to an increase
in the metallothionein
fraction.
Settlemire and Matrone (3) have proposed a possible
mechanism by which a high zinc intake could decrease
hepatic iron concentration.
They contend that the decreased tissue iron results from inhibition of iron incorporation
into ferritin, or its increased release from
ferritin, or both, rather than an increased mobilization
of ferritin iron. They found that 94% of the decrease in
hepatic iron in the rats fed the high zinc diet could be
accounted for by decrease in the ferritin fraction.
Hepatic copper content was not altered by the
amounts of dietary zinc we used. Cox and Harris (2)
reported that excess zinc in the diet causes an early and
marked loss of hepatic iron as well as a significant decrease in hepatic copper. This phenomenon occurs four
weeks after the loss of hepatic iron. Their rats also developed an anemia. In the present study zinc-supplemented rats had normal hematocrit
values and there
was no significant decrease in hepatic copper. This may
be attributed to subtoxic amounts of dietary zinc or that
we did not allow enough time for such a reduction to
become evident.
We thank Mr. Richard
tance.
Harnish
(UCLA) for his technical
usia-
References
1. Smith, S. E., and Larson, E. J., Zinc toxicity in rate. Antagonistic
effects of copper and liver. J. Biol. Chem. 163,29 (1946).
2. Cox, D. H., and Harris, D. L, Effects of excessdietary zinc on iron
and copper in the rat. J. Nutr. 70,514 (1960).
3. Settlemire, C. T., and Matrone, G., In vivo interference of zinc with
ferritin iron in the rat. J. Nutr. 92, 153 (1967).
4. Pallauf, J., and Kirchgessner, M., Zinc status in depletion and repletion and its relation to vitamins and trace elements. In Trace Element Metabolism in Animals, 2. W. G. Hoekstra, Ed., University
Park Press, Baltimore, Md., 1974, pp 534-537.
5. Prasad, S. D., Oberleas, D., Wolf, P., and Horwitz, J. P., Studies
on zinc deficiency: Changes in trace elements and enzyme activities
in tissues of zinc-deficient rats. J. Clin. Invest. 46,549 (1967).
6. Prasad, A. S., and Oberleas, D., Biochemical
effects of zinc deficiency in experimental
animals. In Clinical Application
of Zinc
Metabolism, W. J. Pories, Ed. C. C Thomas, Springfield, 111.1974, pp
19-42.
7. Todd, W. R., Elvehjem, C. A., and Hart, E. B., Zinc in the nutrition
of the rat. Am. J. Physiol. 107,146 (1934).
S. Day, H. G., and McCollusn,
E. V., Effect of acute dietary zinc deficiency in the rat. Proc. Soc. Exp. Biol. Med. 45, 282 (1940).
9. Macapinlac, M. P., Pearson, W. N., and Darby, W. J., Some characteristics of zinc deficiency in the albino rat. In Zinc Metabolism,
A. S. Prasad, Ed., C. C Thomas, Springfield, III., 1966.
10. Dixon, W. J., and Massey, F. J., Introduction
to Statistical
Analysis, McGraw-Hill, Inc., New York, N. Y., 1969, p 204.
11. Edmud, J. F., and Griffith, J. Q.,The Rat in Laboratory Investigation. Hafner Pub. Co., Inc., New York, N. Y., 1967, p 411.
12. Chen, R. W., Eakin, D. J., and Whanger, P. D., Biological function
of met.allothionein. HIts role in zinc metabolism in the rat Nutr. Rep.
mt. 10, 195 (1974).
CLINICALCHEMISTRY,
Vol. 23, No. 10, 1977 1837