Effect of dietary calcium on growth, calcium, magnesium and phosphorus contents and fatty acid composition of individual tissues in rats


Yasuda, M.; Okamura, Y.; Yoshikawa, M.; Takeeda, T.; Nakamoto, Y.; Morisaka, K.

Chemical and Pharmaceutical Bulletin 31(11): 4152-4156

1983


In order to determine the effects of diet containing three calcium (Ca) levels (none, 0.8 or 2.4%) on rats, the Ca, magnesium and phosphorus contents, fatty acid composition in tissues as well as their growth were examined. In the groups fed with Cα-free and 2.4% Ca diet the growth of rats was suppressed compared with that of a control group (0.8% Ca). In both groups the relative weight of brain with respect to the body weight increased and in the group fed with 2.4% Ca diet, the weight of kidney increased both relatively and absolutely. Ca content in all tissues except the kidney of female rats decreased to various extents in the group fed with Cα-free diet. In the group fed with 2.4% Ca diet, the Ca content increased markedly only in the kidney. In the group fed with 2.4% Ca diet, magnesium content decreased only in bone, and phosphorus content was not affected by any dietary Ca level. Fatty acid compositions of the heart, liver and kidney changed as dietary Ca levels were varied, but no change was observed in the brain. Namely, in the group fed with 2.4% Ca diet, oleic and eicosatrienoic acids increased, but linoleic and arachidonic acids decreased. On the other hand, consistent changes of composition in the Cα-free diet group were not observed in any individual tissue of either sex. From these results, it was concluded that the growth of rats was suppressed in the groups fed not only with Cα-free but also high Ca level diets and that the mineral content and fatty acid composition in various tissues changed as the dietary Ca level was varied.

4152
Vol.
31
(1983)
[
Chem.
Pharm.
Bull.]
31(11)4152-4156(1983)
Effect
of
Dietary
Calcium
on
Growth,
Calcium,
Magnesium
and
Phosphorus
Contents
and
Fatty
Acid
Composition
of
Individual
Tissues
in
Rats
MASAHIDE
YASUDA,*'
YOSHIHIDE
OKAMURA,
b
MASAHIKO
YOSHIKAWA,
b
TOSHIYUKI
TAKEEDA,a
YASUO
NAKAMOTOa
and
KATSUAKI
MORISAKAa
Osaka
College
of
Pharmacy,"
2-10-65
Kawai,
Matsubara,
Osaka
580,
Japan
and
Taihei
Kagaku
Sangyo,
Co.,
Ltd.,"
18
Koraihashizume-cho,
Higashi-ku,
Osaka
541,
Japan
(Received
February
18,
1983)
In
order
to
determine
the
effects
of
diet
containing
three
calcium
(Ca)
levels
(none,
0.8
or
2.4%)
on
rats,
the
Ca,
magnesium
and
phosphorus
contents,
fatty
acid
composition
in
tissues
as
well
as
their
growth
were
examined.
In
the
groups
fed
with
Ca-free
and
2.4%
Ca
diet
the
growth
of
rats
was
suppressed
compared
with
that
of
a
control
group
(0.8%
Ca).
In
both
groups
the
relative
weight
of
brain
with
respect
to
the
body
weight
increased
and
in
the
group
fed
with
2.4%
Ca
diet,
the
weight
of
kidney
increased
both
relatively
and
absolutely.
Ca
content
in
all
tissues
except
the
kidney
of
female
rats
decreased
to
various
extents
in
the
group
fed
with
Ca-free
diet.
In
the
group
fed
with
2.4%
Ca
diet,
the
Ca
content
increased
markedly
only
in
the
kidney.
In
the
group
fed
with
2.4%
Ca
diet,
magnesium
content
decreased
only
in
bone,
and
phosphorus
content
was
not
affected
by
any
dietary
Ca
level.
Fatty
acid
compositions
of
the
heart,
liver
and
kidney
changed
as
dietary
Ca
levels
were
varied,
but
no
change
was
observed
in
the
brain.
Namely,
in
the
group
fed
with
2.4%
Ca
diet,
oleic
and
eicosatrienoic
acids
increased,
but
linoleic
and
arachidonic
acids
decreased.
On
the
other
hand,
consistent
changes
of
composition
in
the
Ca-free
diet
group
were
not
observed
in
any
individual
tissue
of
either
sex.
From
these
results,
it
was
concluded
that
the
growth
of
rats
was
suppressed
in
the
groups
fed
not
only
with
Ca-free
but
also
high
Ca
level
diets
and
that
the
mineral
content
and
fatty
acid
composition
in
various
tissues
changed
as
the
dietary
Ca
level
was
varied.
Keywords—dietary
calcium;
growth
of
rat;
tissue;
calcium;
magnesium;
phosphorus;
fatty
acid
composition
It
is
currently
believed
that
one
cause
of
bone
fracture
of
children
may
be
a
deficiency
of
calcium
(Ca)
intake
due
to
changes
of
dietary
intake,
and
it
is
recommended
that
more
Ca
should
be
taken
in
the
diet
and/or
nutrient
supplements
in
childhood.
Diet
is
also
important
in
biological
experiments'''
)
and
the
benefits
of
using
semisynthetic
or
semipurified
diet
in
toxicological
and/or
biochemical
experiments
have
been
discussed.
3)
It
is
particularly
important
to
use
semipurified
diets
in
studies
where
an
essential
nutrient
is
used.
4)
In
the
present
work,
our
purpose
was
to
determine
the
effects
of
dietary
Ca
levels
on
growth,
mineral
content
and
fatty
acid
composition
of
lipid
in
individual
tissues
of
rats
given
semipurified
diets.
Materials
and
Methods
Experimental
Diets
Although
nutrient
forms
of
Ca
include
Ca
phosphate,
carbonate
and
lactate
salts,
CaHPO
4
was
selected
as
the
Ca
source
for
nutrient
evaluation.
Three
different
Ca
contents
in
powdered
and
semipurified
diets
were
used
as
shown
in
Table
I.
Animals
and
Feeding
SPF
Fischer
344/Nslc
male
and
female
rats,
25
d
old,
were
kept
in
plastic
cages
with
sterilized
Beta
Chips
(Charles
River
Japan,
Inc.).
After
a
preliminary
feeding
with
a
sterilized
stock
diet
(Type
NMF,
No.
11
4153
TABLE
I.
Percentage
Composition
of
Diets"
)
Ingredient
Ca-free
Control'
)
2.4%
Ca
Casein,
vitamin-free
23.0
23.0
23.0
Sucrose
64.6
64.6
58.0
Vegetable
oil'
)
5.0
5.0
5.0
Vitamin
mix"
)
1.0
1.0
1.0
Mineral
mix'
)
3.0
3.0
3.0
Na
2
HPO
4
3.4
CaHPO
4
.
3.4
10.0
a)
The
diets
were
prepared
by
Oriental
Yeast
Co.,
Ltd.,
Japan.
b)
Control
diet
contained
0.8%
calcium.
c)
The
percentage
fatty
acid
composition
of
vegetable
oil
was
myristic
(2.6%),
palmitic
(32.6%),
stearic
(11.0%),
oleic
(37.3%),
linoleic
(11.8%),
linolenic
(1.3%)
and
octadecatetraenoic
plus
eicosamonoenoic
(3.4%)
acids.
d)
Vitamin
mix
provided
the
following
in
mg/kg
diet:
ot-tocopherol,
50;
menadione,
52;
thiamine
HCl,
12;
riboflavine,
40;
pyridoxine,
8;
cyanocobalamin,
0.005;
ascorbic
acid,
300;
biotin,
0.2;
folic
acid,
2;
Ca
pantothenate,
50;
p-aminobenzoic
acid,
50;
niacin,
60;
inositol,
60;
choline
chloride,
2000;
retinyl
acetate
(500
I.U.);
ergocarciferol,
(1000
I.U.).
e)
Mineral
mix
provided
the
following
in
g/kg
diet:
magnesium
chloride,
6.0;
potassium
citrate,
6.21;
potassium
chloride,
5.91;
sodium
chloride,
2.46;
potassium
sulfate,
0.63;
ferric
citrate,
0.48;
potassium
iodide,
0.012;
sodium
fluoride,
0.012;
manganous
sulfate,
0.006;
potassium
aluminate,
0.012;
sodium
selenate,
0.012;
Cupric
sulfate
5H
2
0,
0.012;
cobalt
chloride
6H
2
0,
0.012;
zinc
sulfate
7H
2
0,
0.012.
175
-
150
-
125
-
100
-
ir
75
_
k
s
,
Fig.
1.
Growth
Curves
of
Fischer
344
Rats
Fed
Semipurified
Diets
Containing
Three
Levels
of
Calcium
for
6
Weeks
50
Vertical
lines
indicate
the
standard
error
of
the
mean.
0=0,
calcium-free
diet;
P=P,
control
diet;
-
,
2.4%
calcium
diet.
,
male
rats;
0
1
2
3
Week
4
5
6
female
rats.
Oriental
Yeast,
Co.,
Ltd.,
Japan),
the
rats
were
divided
into
3
groups
of
10
animals
and
fed
with
an
experimental
diet
from
4
weeks
of
age.
Both
diet
and
deionized
water
were
allowed
ad
libitum
for
6
weeks.
The
animal
room
was
maintained
at
24
°C,
55%
relative
humidity,
18
air
changes
per
hour,
and
with
a
12
h
(0600
to
1800
h)
light-dark
cycle.
After
the
experimental
period
of
6
weeks,
each
rat
was
exsanguinated
by
decapitation
and
the
brain,
heart,
liver,
kidney
and
femur
were
taken
out
and
weighed
immediately.
All
tissues
were
frozen
and
stored
at
-20
°C
until
analyzed.
Analytical
Methods
After
digestion
of
tissues
with
nitric
acid
and
hydrogen
peroxide,
Ca
and
magnesium
were
estimated
by
the
use
of
an
atomic
absorption
spectrophotometer
(Hitachi,
type
207)
in
1.0%
LiCl
3
solution
(final
concentration)
to
exclude
the
interference
of
phosphate,
and
total
phosphorus
was
assayed
colorimetrically
by
the
method
of
Bartlett.
5)
Fatty
acid
compositions
of
various
tissues
except
the
femur
were
measured
by
a
gas
liquid
chromatography
method
as
described
in
our
previous
paper.
6)
Results
and
Discussion
Growth
and
Mineral
Contents
in
Organs
As
shown
in
Fig.
1,
male
and
female
rats
fed
with
a
control
diet
(0.8%
Ca)
grew
normally.
However,
rats
of
both
sexes
fed
with
Ca-free
and
2.4%
Ca
diets
grew
little,
and
body
weights
were
significantly
lower
than
in
the
control
group
over
the
experimental
period.
The
transient
Bo
dy
we
ig
ht
(g
)
4154
Vol.
31
(1983)
loss
in
body
weight
observed
at
the
beginning
of
the
experiment
may
be
attributed
to
reduced
food
intake
due
to
the
unfamiliar
taste.
In
the
control
group,
the
food
intake
of
males
gradually
increased
from
about
4
g
at
the
3rd
day
to
7
g
at
the
25th
day
after
the
beginning
of
the
experiment
and
was
9
g
at
the
end
of
the
experiment.
On
the
other
hand,
the
food
intake
of
females
was
somewhat
less
than
that
of
males
over
the
experimental
period.
No
apparent
difference
in
food
intake
among
the
groups
was
observed
during
the
first
25
d,
but
the
food
intake
of
rats
fed
with
a
Ca-free
or
2.4%
Ca
diet
scarcely
increased
during
the
subsequent
part
of
the
experimental
period.
The
absolute
and
relative
organ
weights
of
male
rats
fed
the
control
diet
were
as
follows
(in
grams
and
ratio
to
body
weight):
brain,
1.28
+
0.04
and
0.73
+
0.04;
heart,
0.59
+
0.06
and
0.38
±
0.01;
liver,
8.40
±
0.23
and
4.64
±
0.14;
kidney,
1.77
±
0.04
and
1.00
±
0.02;
bone,
0.68
+
0.01
and
0.39
+
0.02.
There
was
no
difference
in
these
values
between
males
and
females,
except
for
the
kidney.
The
absolute
kidney
weights
of
male
and
female
rats
were
1.13
+
0.05
and
1.02
+
0.01
g
in
the
Ca-free
diet
group,
1.77
+
0.04
and
1.39
+
0.04
g
in
the
control
group
and
1.94
+
0.03
and
1.45
+
0.05
g
in
the
2.4%
Ca
diet
group,
respectively.
The
relative
kidney
weights
of
male
and
female
rats
increased
about
100
and
50%,
respectively,
in
the
group
fed
the
2.4%
Ca
diet.
In
the
groups
fed
with
Ca-free
and
2.4%
Ca
diet,
the
relative
weight
of
the
brain
in
males
and
females
increased
about
70
and
40%,
but
the
absolute
weight
was
not
affected.
The
absolute
and
relative
weights
of
the
other
organs
did
not
change
in
male
and
female
rats
fed
with
any
of
the
diets.
Next,
we
determined
the
effects
of
dietary
Ca
on
the
concentrations
of
minerals
in
the
organs.
Ca
and
magnesium
contents
in
organs
of
male
rats
fed
the
control
diet
for
6
weeks
were
48.9
+
4.33
and
156
+
7.81
µgig
in
brain,
33.3
+
5.35
and
193
+
7.43
µgig
in
heart,
39.6
±
7.85
and
206
±
4.59
µgig
in
liver,
76.8
±
3.90
and'
183
±
5.35
itg/g
in
kidney,
and
230
±
6.50
and
4.04
±
0.06
mg/g
in
bone,
respectively,
and
there
was
no
significant
sex
difference.
In
the
group
fed
the
Ca-free
diet,
the
Ca
content
in
all
organs,
except
the
kidney
of
females,
decreased
to
various
extents,
while
the
Ca
content
in
the
kidney
of
females
increased
about
3.5
times.
In
the
group
fed
the
2.4%
Ca
diet,
the
Ca
content
of
males
and
females
increased
about
60
and
50
times,
respectively,
as
compared
with
the
control
group.
In
the
group
fed
the
2.4%
Ca
diet,
the
magnesium
content
decreased
only
in
bone,
by
about
45
and
20%
in
males
and
females,
respectively.
On
the
other
hand,
total
phosphorus
contents
in
brain,
heart,
liver,
kidney
and
bone
of
male
rats
of
the
control
group
were
3.70
+
0.13,
3.04
+
0.02,
3.25
+
0.14,
3.72
+
0.35
and
124
+
0.51
mg/g
of
tissue,
respectively,
and
these
values
were
independent
of
sex
and
dietary
Ca
level.
The
findings
in
this
study
suggest
that
immoderate
consumption
of
Ca
results
in
suppression
of
growth
and
alteration
of
mineral
contents
in
organs
such
as
kidney
and
bone.
This
may
have
important
implications
for
human
nutrition.
Fatty
Acid
Composition
of
Organs
In
experiments
with
rabbits
and
rats,
it
was
shown
that
plasma
lipid
levels
decreased
and
tissue
lipid
levels
varied
as
dietary
Ca
level
was
increased.'
-9)
These
findings
suggest
that
lipid
mobilization
among
tissues
and
lipid
metabolism
are
affected
by
dietary
Ca
levels,
and
consequently
the
fatty
acid
composition
of
tissue
lipid
is
also
affected.
In
this
study,
we
observed
the
effects
of
diets
containing
three
Ca
levels
on
the
fatty
acid
composition
of
tissue
lipid
(Table
II).
The
composition
of
brain
lipid
was
little
affected
by
dietary
Ca
level.
However,
the
fatty
acid
compositions
of
heart,
liver
and
kidney
of
males
and
females
changed
as
the
dietary
Ca
level
varied.
Namely,
in
the
group
fed
the
2.4%
Ca
diet,
oleic
and
eicosatrienoic
acids
increased,
but
linoleic
and
arachidonic
acids
decreased.
However,
no
consistent
changes
of
the
compositions
in
the
Ca-free
diet
group
were
observed
among
tissues,
and
there
were
no
sex
differences
in
the
compositions.
No.
11
4155
TABLE
II.
Fatty
Acid
Composition
in
Individual
Tissues
from
Rats
Fatty
acids
Brain
Heart
Liver
Kidney
Male
Female
Male
Female
Male
Female
Male
Female
2.4+0.1
2.6+0.1
2.6+0.2
2.9+0.1
1.9+0.1
1.7+0.1
C
16
:
ald
2.2+0.1
2.6+0.1 1.7+0.2
2.2+0.1
1.7+0.1
1.8+0.2
2.5+0.1
2.6+0.1
2.1+0.1
1.9+0.1
1.8+0.1
1.9+0.2
22.4+0.5
21.2+0.1
14.5+0.6
b)
13.8+0.4
20.4+0.5`
)
21.1+0.2
18.8+0.7a
)
17.7+0.4°
C
16:0
23.5+0.6
21.7+0.5
12.5+0.2
14.0+0.4
23.3+0.5
20.1+0.3
21.1+0.7
20.4+0.9
23.6+0.9
21.7+0.1
13.7+0.7
14.4+0.5
20.6+0.6
1
'
)
21.9+0.7
17.7+0.0
18.1+0.6
1.6+0.2
1.6+0.2
3.8+0.2
3.7+0.1
2.7+0.3
3.5+0.3
C
16:1
2.2+0.3
2.2+0.1
3.7+0.4
4.5+0.1
3.8+0.2
4.4+0.2
2.2+0.1
2.3+0.2
4.2+0.2
4.6+0.1
3.8+0.1
4.0+0.4
4.2+0.2
4.3+0.2
C
18
:
ald
4.4+0.1
4.7+0.2
4.2+0.1
4.3+0.1
23.3+0.1
23.8+0.3
21.5+0.5
20.7+0.3
17.4+0.8
15.9+0.2
d)
18.8+0.4
18.2+0.5
C
18:0
22.8+0.9
22.9+0.6
21.8+0.4
21.0+0.5
15.7+0.8
20.5+0.2
18.3+0.1
17.9+0.1
22.9+1.0
23.6+0.2
21.1+0.2
20.5+0.6
15.6+0.5
14.4+0.4
d)
18.3+0.5
18.9+0.7
19.5+0.7
20.3+0.9
17.3+0.6
20.2+0.5
26.8+0.9°
33.3+0.2
d)
20.2+0.7
23.6+0.9
C
18:1
19.7+0.8
19.4+0.5
17.2+0.4
19.9+0.4
30.5+0.6
24.8+0.1
20.0+0.3
23.6+0.4
18.7+0.5
20.6+0.1
20.9+0.1
d)
23.9+0.8'
)
32.5+0.4"
)
36.7+0.8
d)
26.9+0.3
d)
29.5+0.5
d)
8.3+0.4
d)
7.9+0.6°
4.3+0.3
2.5+0.1
6.7+0.5
5.4+0.3
C
18:2
15.1+0.1
10.8+0.5
3.4+0.1
2.1+0.4
6.2+0.1
4.9+0.2
7.6+0.4
d)
6.6+0.0
2.4+0.2a
)
1.4+0.3
2.4+0.2
d)
2.3+0.4
d)
C
18:4
1.2+0.1
1.2+0.1
plus
1.2+0.2
1.5+0.1
C
20:1
1.1+0.1
1.3+0.2
2.4+0.4
d)
3.7+0.6
4.6+0.5
8.9+0.1
3.1+0.2
4.8+0.4
C
20:3
7.1+0.3
3.7+0.3
5.7+0.4
8.0+0.4
5.2+0.3
3.7+0.4
6.7+0.3
8.2+0.4
d)
10.9+0.4
d)
11.2+0.6°
7.3+0.1°
8.3+0.1
d)
11.2+0.3
10.3+0.3
22.3+0.6
d)
21.5+0.2
15.8+0.5"
)
9.9+0.1
d)
24.1+0.7
0
20.2+0.7
C
20:4
10.1+0.3
10.3+0.6
17.8+0.5
20.3+0.3
12.9+0.6
14.2+0.1
20.5+0.5
20.0+0.6
10.4+0.2
9.9+0.1
18.1+0.1
16.8+0.2
d)
8.6+0.3
d)
6.0+0.3
d)
17.9+0.3
d)
15.0+0.3
d)
3.2+0.2
2.7+0.2
C
22:4
2.2+0.2
3.4+0.3
2.8+0.1
2.4+0.1
1.5+0.1
1.5+0.2
1.1+0.1
1.0+0.0
1.3+0.3
1.0+0.1
C
22:5
1.3+0.1
1.8+0.3
1.8+0.1
2.8+0.2
1.4+0.1
1.4+0.2
1.1+0.2
1.2+0.2
1.4+0.1
1.1+0.2
1.0+0.1
1.0+0.3
12.5+0.1
13.5+0.2
7.6+0.4
d)
6.6+0.2
d)
4.9
±
0.4
b)
3.3+0.4
1.8+0.1
1.6+0.2
C
22:6
11.8+0.5
12.4+0.4
3.0+0.4
3.7+0.1
2.9+0.3
3.4+0.1
1.5+0.1
1.2+0.2
11.4+1.0
12.3+0.3
5.5
+0.2c
)
4.2+0.4
3.4+0.3
2.9+0.2
1.0+0.1
1.0+0.5
Minus
signs
indicate
trace.
Values
are
the
mean
+
S.E.
of
percentages
of
fatty
acids
in
4
experiments.
Values
in
the
upper,
middle
and
lower
rows
are
for
the
calcium-free,
control
and
2.4%
calcium
diet
groups,
respectively.
Significant
differences
from
the
control
groups
are
as
follows:
a)
p
<
0.05;
b)
p
<
0.02;
c)
p
<0.01;
d)
p
<0.001.
One
of
the
major
functions
of
fatty
acid
moieties
of
tissue
lipid
as
phospholipids
is
to
contribute
to
the
structure
and
function
of
cell
membranes.'
o,11)
It
is
also
generally
accepted
that
Ca
is
important
for
regulating
the
permeability
and
integrity
of
cell
membranes.
The
fact
4156
Vol.
31
(1983)
that
the
fatty
acid
composition
of
tissue
lipid
changed
as
dietary
Ca
level
was
varied
suggests
that
dietary
Ca
may
affect
metabolic
transport
and
function
in
organs.
References
1)
P.
M.
Newberne,
J.
G.
Bieri,
G.
M.
Briggs
and
M.
C.
Nesheim,
ILAR
News,
XXI
(2),
A3
(1978).
2)
M.
J.
L.
Clapp,
Laboratory
Animals,
14,
253
(1980).
3)
I.
Knudsen
and
0.
A.
Meyer,
Toxicology,
4,
203
(1975).
4)
P.
M.
Newberne
and
J.
G.
Fox,
Laboratory
Animal
Sci.,
30,
352
(1980).
5)
G.
R.
Bartlett,
J.
Biol.
Chem.,
234,
466
(1959).
6)
T.
Fujita
and
M.
Yasuda,
Jpn.
J.
Pharmacol.,
23,
899
(1973).
7)
R.
M.
Dougherty
and
J.
M.
Iacono,
J.
Nutr.,
109,
1934
(1979).
8)
J.
M.
Iacono,
J.
Nutr.,
104,
1165
(1974).
9)
R.
D.
Pace,
J.
W.
Chen
and
J.
Protho,
Nutr.
Rep.
Inc.,
7,
121
(1973).
10)
J.
B.
Finean,
"Form
and
Function
of
Phospholipids,"
ed.
by
G.
B.
Ansell,
J.
N.
Hawthorne
and
R.
M.
C.
Dawson,
Elsevier
Scientific
Publishing
Company,
New
York,
1973,
pp.
171-203.
11)
W.
C.
McMurray,
"Form
and
Function
of
Phospholipids,"
ed.
by
G.
B.
Ansell,
J.
N.
Hawthorne
and
R.
M.
C.
Dawson,
Elsevier
Scientific
Publishing
Company,
New
York,
1973,
pp.
205-251.