Food and Nutrition Sciences, 2010, 1, 13-18
doi:10.4236/fns.2010.11003 Published Online July 2010 (http://www.SciRP.org/journal/fns)
13
Effect of Calcium and Phosphorus on Nonhaeme
Iron Absorption and Haematogenic
Characteristics in Rats
Heba Ezz El-Din Yossef
Nutrition and Food Science Department, Faculty of Home Economics, Minufiya University, Shebin El Kom, Egypt.
Received June 3rd, 2010; revised July 3rd, 2010; accepted July 7th, 2010.
ABSTRACT
The objectives of this study were to use the dry thyme leaves as source of nonhaeme iron and evaluate the effects of cal-
cium, phosphorus and calcium + phosphorus on nonhaeme iron absorption and haematogenic characteristics in rats.
Thirty adult male albino ra ts, weighing 150 ± 5 g were divided into five groups. The first group fed basal diet, the sec-
ond group fed thyme diet, the third group fed thyme diet + calcium, the fourth group fed thyme diet + phosphorus and
the fifth group fed thyme diet + calcium + phosphorus. All groups fed experimental diets for six weeks. Hemoglobin
(Hb), haematocrit (Ht), red blood cell (RBC), mean corpuscular volume (MCV), serum iron (SI), serum ferritin (SF),
total iron–binding capacity and transferrin saturation were determined at the beginning and the at end of the experi-
ment. Iron in diet, Fe intake, Fe feces and Fe absorption were also evaluated. The results indicated that the lowest Fe
absorption was observed in rats fed the thyme diet + calcium and thyme diet + calcium + phosphorus. Supplementation
the thyme diet with calcium or calcium + phosphorus decreased the values of Hb, Ht, RBC, SI and SF. However, sup-
plementation the thyme diet with phosphorus did not affect in Ht, RBC and MCV but Hb, SI and SF increased. The re-
sults suggest that supp lementation the diet with calcium or calcium + phosphorus interfere with iron absorption.
Keywords: Nonhaeme Iron, Thyme, Iron Absorption, Hemoglobin
1. Introduction
The most common nutritional deficiencies now affecting
all ages involve iron and calcium [1]. In recent years,
Ca-enriched foods have come to be a habitual part of
daily diet [2]. On the other hand, Fe deficiency is the
most common nutritional disorder worldwide, affecting
people of all ages in the both industrialized and devel-
opi93ng countries [3]. Nutritional Fe deficiency arises
when physiological requirement cannot be met by Fe
absorption from the diet. The efficiency of iron absorp-
tion depends on the both bioavailability of dietary iron
and iron status. Iron absorption is influenced by many
factors. Body need, vitamin C, protein and carbohydrate
intakes enhance absorption [4,5]. On the other side,
binding agents such as phytate, oxalate and phosphate,
dietary fiber, calcium, coffee, tea and gastrointestinal
diseases inhabit iron absorption [6-8].
Several studies with animals have clearly shown that
Ca interferes with dietary absorption of Fe and that addi-
tion of Ca to the diet may even induce Fe deficiency [9].
Increased Ca supplementation may have an adverse ef-
fect on the metabolism of some micronutrients such as
iron and zinc [10].
Thyme is source of protein and iron [11]. The iron
content in dry thyme leaves was 117.2 mg/100g dry mat-
ter [12]. It is increasingly recognized that simultaneous
provision of iron, calcium, phosphorous in supplements
may decrease benefit of one or three. These complex
micronutrient interactions and their implications for nu-
tritional interventions are incompletely understood. The
absorption and bioavailability of nonheme iron have not
been adequately studied. Therefore, the objectives of this
study were to use the dry thyme leaves as source of non-
heme iron and evaluate the effects of calcium, phospho-
rus and calcium + phosphorus on nonhaeme iron absorp-
tion and haematogenic characteristics in rats.
2. Materials and Methods
Dry thyme leaves used in this study was purchased from
Shibin El-Kom, Egypt. The thyme leaves were ground,
sieved and stored at – 4°C until use. Calcium carbonate,

Effect of Calcium and Phosphorus on Nonhaeme Iron Absorption and Haematogenic Characteristics in Rats 14
calcium phosphate and sodium phosphate mono hydro-
gen were obtained from Gomhouria Company, Cairo,
Egypt.
2.1 Experimental Design
Thirty adult male albino rats, Sprague drawly strain,
weighing 150 ± 5 g were purchased from Helwan farm.
The rats were housed individually in cage and fed basal
diet for one week for adaptation. The basal diet consisted
of 100 g/kg corn oil; 126.3 g/kg casein; 40 g/kg mineral
mixture, USP XIV; 10 g/kg vitamin mixture; 3 g/kg
DL-methionine and 2 g/kg choline chloride and 50 g/kg
fiber and corn starch 668.7 g/kg [13].
At the beginning of experiment, A 5 ml blood sample
were taken to determine hemoglobin, haematocrit serum
iron, serum ferritin, red blood cell, and total iron-binding
capacity. As the data obtained basis, the rats were di-
vided into five groups, 6 rats per group. The first (control
group) fed basal diet, the second group fed thyme diet
(21 g dry thyme leaves/kg basal diet), the third group fed
thyme diet + double amount of the recommended dietary
allowance of Ca (10 g/kg diet) from CaCO3, the fourth
group fed thyme diet + double amount of the recom-
mended dietary allowance of P (8 g/kg diet) from sodium
phosphate mono hydrogen (Na H PO4) and the fifth
group fed thyme diet + double amount of the recom-
mended dietary allowance of Ca and P from calcium
phosphate (Ca PO4) as described by [14]. Feed intake
was recorded daily. Faces were collected of each animal
daily. Body weight was recorded at the beginning and at
the end of experimental period. At the end of experimen-
tal period (6 weeks), the rats fasted overnight and were
anaesthetized. Blood sample were collected and aliquots
were analyzed to measure the hematological parameters.
The remaining blood was centrifuged to obtain serum for
determination serum iron, serum ferritin and total iron
binding capacity.
2.2 Analytical Methods
Total nitrogen content, crude fiber, fat, moisture, and ash
were determined according to [15]. The carbohydrate
was calculated by difference. The concentration of Fe in
the diets and faces were determined by atomic absorption
spectrophotometer (Perkin Elmer 1100B, Norwalk, and
Ct, USA). Hemoglobin (Hb) red blood cell (RBC) and
haematocrit (Ht) in heparinized blood samples were
measured using automated hematology analyzer (Sysmex,
Kobe, Japan). Total iron-binding capacity (TIBC), serum
iron and serum ferritin levels were determined calorimet-
rically and enzymatically, using sigma diagnostics iron,
ferritin and TIBC reagents, (sigma diagnostics, st. Louis,
MI, USA). Transferrin saturation (%) was calculated
using the following equation: Transferring saturation (%)
= (Serum iron concentration ÷ TIBC) × 100. Mean cor-
puscular volume was calculated as described by [16]
using the following equation:
HT
MCV = RBC × 10
2.3 Statistical Analysis
The experimental data were subjected to an analysis of
variance (ANOVA) for a completely randomized design
using a statistical analysis system [17]. Duncan’s multi-
ple range tests were used to determine the differences
among means at the level of 95%.
3. Result and Discussion
The proximate chemical composition and iron content of
dry thyme leaves were presented in Table 1. Data
showed that the protein (20.5%), carbohydrate (45.2%),
fiber (12.6%) and iron content (122.7 mg/g dry matter)
were high in dry thyme while moisture (4.95%) and fat
(4.6%) were low. These results are agreement with [12]
who reported that the dry thyme was high content in pro-
tein (18.9g/100 dry matter), carbohydrate (49.6g/100g),
fiber (15g/100g dry matter) and iron (117.2mg/100 dry
matter) but low in moisture and fat.
Data in Table 2 showed that rats fed basal diet had the
Table 1. Proximate chemical composition and iron content of dry thyme leaves
Moisture (%) Protein (%) Fat (%) Carbohydrate (%) Fiber (%) Ash (%) Fe (mg/100g)
4.95 ± 0.25 20.5 ± 1.2 4.6 ± 0.36 45.2 ± 0.56 12.6 ± 0.81 12.6 ± 0.81 122.7
Table 2. Fe diet, Fe intake, Fe feces and Fe absorption in rats fed basal diet, thyme diet and thyme diet supplemented with
minerals
Diet Fe diet (mg/kg) Fe intake (mg/kg) Fe feces (mg/kg) Fe absorption (%)
Basal diet 46b ± 1.0 32.03b + 1.74 15.90c + 1.40 50.35a + 1.73
Thyme diet 70.67a ± 2.1 48.48a + 2.42 30.43b + 0.98 37.10b + 1.50
Thyme diet + Ca 69a ± 1 48.55a + 1.61 33.57a + 1.80 31.25c + 1.98
Thyme diet + P 68.67a ±1.53 46.76a + 1.36 29.33b + 1.03 37.63b + 1.32
Thyme diet + Ca + P 70a ± 1.0 48.90a + 1.46 33.68a + 1.70 31.15c + 1.51
LSD 2.53 3.2 2.48 2.95
Means in the same column with different letters are significantly different (p < 0.05)

Effect of Calcium and Phosphorus on Nonhaeme Iron Absorption and Haematogenic Characteristics in Rats 15
lowest (P ≤ 0.05) iron diet and iron intake as compared to
rats fed thyme diet and thyme diet supplemented with
minerals. There was no significantly (P > 0.05) differ-
ence in Fe intake between rats fed thyme diet and rats fed
thyme diet supplemented with minerals. Rats fed basal
diet had lower (P ≤ 0.05) Fe feces and higher Fe absorp-
tion than those fed thyme diet and thyme diet supple-
mented with minerals. The lowest Fe absorption was
observed in rats fed the thyme diet + calcium and thyme
diet + calcium + phosphorus. Although the Fe intake was
lower (P ≤ 0.05) in rats fed basal diet than those fed
thyme diet and thyme diet supplemented with minerals,
the Fe absorption was the highest (P ≤ 0.05). This is due
to the low Fe fecal excretion in rats fed basal diet and
polyphenol compound in thyme diet which had adverse
effect on Fe absorption. These results are agreement with
those reported by [18,19] they reported that polyphenols
inhibited the absorption of nonheme iron. As suggested
by [20] the percentage of iron absorbed decreases as iron
intake increases. Similar results were reported by [21]
who found in humans that those fed bread fortified with
increasing amounts of iron (1, 3 and 5 mg) had lower
percentages of iron absorbed, but their absolute absorption
increased in response to increasing iron intakes.
Fe absorption was lower (P ≤ 0.05) in rats fed thyme
diet + calcium and thyme diet + calcium + phosphorus
than those fed thyme diet and thyme diet + phosphorus.
This may be due to the losses of Fe in feces and the
presence of calcium or and phosphorus in the diet which
inhibit nonhaeme iron absorption. Similar results were
reported by [22,23]. However these results were differed
from those reported by [2,24] they found that feeding rats
high calcium diet for two weeks do not inhibit iron ab-
sorption.
Effect of calcium and phosphorous on the hemoglobin
(Hb), haematocrit (Ht), red blood cell (RBC) and mean
corpuscular volume(MCV) in rat fed basal diet, thyme
diet and thyme diet supplemented with minerals are
shown in Table 3. There was no significant (P > 0.05)
change in Hb between rats fed basal diet. However, Hb
was significantly (P ≤ 0.05) affected in rats fed thyme diet,
thyme diet supplemented with calcium, thyme diet sup-
plemented with phosphorus and thyme diet supplemented
with calcium + phosphorus. Hemoglobin was signifi-
cantly (P ≤ 0.05) increased in rats fed thyme diet and
thyme diet supplemented with phosphorus. However,
hemoglobin was significantly (P ≤ 0.05) decreased in rats
fed thyme diet supplemented with calcium and thyme diet
supplemented with calcium + phosphorus.
Haematocrit and red blood cell did not significantly (P
≤ 0.05) affect in rats fed basal diet, thyme diet and thyme
diet supplemented with phosphorus. However, haema-
tocrit and red blood cell were significantly (P ≤ 0.05)
decreased in rats fed thyme diet supplemented with cal-
cium and thyme diet supplemented with calcium +
phosphorus.
Mean corpuscular volume was significantly (P ≤ 0.05)
decreased in rats fed basal diet and thyme diet. Mean
corpuscular volume was significantly (P ≤ 0.05) in-
creased in rats fed thyme diet supplemented with calcium.
However, mean corpuscular volume did not significantly
(P ≤ 0.05) affect in rats fed thyme diet supplemented
with phosphorus and thyme diet supplemented with cal-
cium + phosphorus.
These data indicated that supplementation the diet with
calcium decreased the values of Hb, Ht and RBC. Sup-
plementation the diet with phosphorus did not affect in
Ht, RBC and MCV but Hb increased. However, supple-
mentation the diet with calcium + phosphorus decreased
the values of Hb, Ht and RBC but MCV did not affect.
Increased Fe intake response with increment of Hb con-
centration [25]. Hemoglobin concentration was negative-
Table 3. Effect of calcium and phosphorous on the he moglobin, haematocrit, red blood cell and mean corpuscular volume in
rat fed basal, thyme diet and thyme diet supplemented with mine r a ls
Basal
Diet LSD Thyme diet LSD Thyme Diet + Ca LSD Thyme Diet + P LSD
Thyme
Diet + Ca +
P
LSD
Hb (g/dl)
Initial
Final
11.9a ± 0.35
12.2a ± 0.34
0.599
11.9b + 0.35
12.45a ± 0.17
0.47
15.2a ± 0.58
10.55b ± 0.87
1.27
11.76b ± 0.23
12.45a ± 0.17
0.35
14.25a ± 1.1
11.9b ± 0.12
1.35
Ht (%)
Initial
Final
33.5a ± 1.7
35.5a ± 0.6
2.2
35.5a ± 0.57
34a ± 1.2
1.57
45a ± 1.2
37b ± 2.3
3.15
38.5a ± 0.58
37.5a ± 0.57
0.998
41.5a ± 2.88
35.5b ± 0.58
3.6
RBC
(mil/cmm)
Initial
Final
3.87a ± 0.2
4.18a ± 0.1
0.32
4.1a ± 0.12
4.05a ± 0.1
0.16
5.1a ± 0.23
3.75b ± 0.64
0.83
4.4a ± 0.35
4.25a ± 0.1
0.43
4.75a ± 0.29
4b ± 0.12
0.38
MCV (fl)
Initial
Final
86.6a ± 1
84.9b ± 0.7
1.53
86.6a + 1
84.31b ± 1.2
1.96
88.2b ± 1.7
98.7a ± 1.8
3.03
87.5a ± 5.6
88.2a ± 0.17
6.85
87.36a ± 0.75
88.75 a ± 1.1
1.63
Hb: Hemoglobin; Ht: Haematocrit; RBC: Red blood cell; MCV: Mean corpuscular volume. Means in the same column for each variable with dif-
ferent letters are significantly different (p < 0.05)

Effect of Calcium and Phosphorus on Nonhaeme Iron Absorption and Haematogenic Characteristics in Rats 16
ly and significantly correlated with the intake of calcium
[26]. Calcium supplementation reduced heme and total
iron without significantly affecting nonheme-iron ab-
sorption [23].
The effect of calcium and phosphorus on the serum
iron (SI), serum ferritin (SF), total iron binding capacity
(TIBC) and transferrin saturation (TS) in rat fed basal
diet, thyme diet and thyme diet supplemented with min-
erals are shown in Table 4. Serum iron, serum ferritin
and transferrin saturation did not significantly (P ≤ 0.05)
affect in rats fed basal diet. Serum iron, serum ferritin and
transferrin saturation were significantly (P ≤ 0.05) in-
creased in rats fed thyme diet and thyme diet supple-
mented with phosphorus. However, SI, SF and TS were
significantly (P ≤ 0.05) decreased in rats fed thyme diet
supplemented with calcium and thyme diet supplemented
with calcium + phosphorus. The improvement in SI, SF
and TS for rats fed thyme diet and thyme diet + P may be
due to low iron stores in these groups.
Similar results were obtained by [27] who reported
that high calcium supplementation at doses 500 and 1000
mg/day for 3 months reduce serum ferritin concentration
in women. In an extensive study in France (n = 1108),
serum ferritin concentration was negatively and signifi-
cantly correlated with the intake of calcium [26]. Similar
findings were made in a study on French students (n = 476)
[28]. Nonheme iron can enhance levels of serum iron and
serum ferritin [29].
Total iron binding capacity was significantly (P ≤ 0.05)
decreased in rats fed thyme diet and thyme diet supple-
mented with phosphorus. Total iron binding capacity did
not significantly (P ≤ 0.05) affect in rats fed basal diet
and thyme diet supplemented with calcium + phosphorus.
However, total iron binding capacity was significantly (P
≤ 0.05) increased in rats fed thyme diet supplemented
with calcium.
The effect of calcium and phosphorus on the feed in-
take, body weight gain and feeding efficiency ratio in rat
fed basal diet; thyme diet and thyme diet supplemented
with minerals are shown in Table 5. There were no sig-
nificant (P > 0.05) changes in feed intake, body weight
gain and feeding efficiency ratio among rats fed basal
diet; thyme diet and thyme diet supplemented with cal-
cium and thyme diet supplemented with calcium and
phosphorus.
This finding was in agreement with [30] who reported
Table 4. Effect of calcium and phosphorous on serum iron, serum ferritin, total iron binding capacity and transferrin saturation in rat fed
basal, thyme diet and thyme diet supplemented with minerals
Basal
Diet LSD Thyme Diet LSD Thyme Diet + Ca LSD Thyme Diet + P LSD Thyme Diet + Ca + P LSD
SI (

g/dl)
Initial
Final
69.5a ± 2.9
73a ± 2.3
4.52
67b ± 2.3
94a ± 1.2
3.16
110a ± 3.5
94b ± 3.6
5.99
102b ± 2.3
114.5a ± 2.8
4.52
99.5a ± 1.7
96b ± 1.2
2.5
SF (

g/dl)
Initial
Final
29.75a ± 1.4
32.05a ± 1.7
2.7
18.6b ± 0.92
23.75a ± 2
2.72
35.15a ± 0.4
24.2b ± 1.5
1.9
26.8b ± 0.92
31.7a ± 1.4
2.04
31.5a ± 2.9
27b ± 1.5
3.98
TIBC
(

g/dl)
Initial
Final
310a ± 17.3
302a ± 15
28
336.5a ± 1.7
305b ± 11.5
14.3
293b ± 6.9
317.5a ± 2.9
9.18
319a ± 11.5
300b ± 5.8
15.79
311a ± 1.2
312.5a ± 2.88
3.8
T.S (%)
Initial
Final
22.42a ± 2.1
24.17a ± 2
3.6
19.91b ± 0.585
30.86a ± 1.54
2
37.54a ± 2
29.61b ± 1.4
3
32.6b ± 0.46
38.2a ± 1.7
2.15
32 a ± 0.44
30.72b ± 0.1
0.55
SI: Serum iron, SF: Serum ferritin, TIBC: Total iron binding capacity, ST: Transferrin saturation. Means in the same column for each variable
with different letters are significantly different (p < 0.05)
Table 5. Feed intake, body weight gain and feeding efficiency ra tio in rat fed basal, thyme diet and thyme diet supplemented
with minerals
Diet Feed intake (g) Body weight gain (g) FER
Basal diet 696a ± 22.6 15.5a ± 2.29 2.23a ± 0.38
Thyme diet 690a ± 25.98 14.5a ± 1.15 2.1a ± 0.1
Thyme diet + Ca 703.5a ± 15.8 15.25a ± 1.56 2.17a ± 0.21
Thyme diet + P 681a ± 10.39 15.25a ± 1.66 2.24a ± 1.32
Thyme diet + Ca + P 698.8a ± 30.34 16.75a ± 1.15 2.4a ± 0.23
LSD 40.41 2.94 0.47
Means in the same column with different letters are significantly different (p < 0.05)

Effect of Calcium and Phosphorus on Nonhaeme Iron Absorption and Haematogenic Characteristics in Rats 17
that there no differences in food intake and body weight
gain among groups fed flours supplemented with reduced
and increased iron.
From the above results, it could be concluded that
supplementation the diets with calcium carbonate (as a
source of Ca) and calcium phosphate (as a source of Ca +
P) reduced the iron absorption in rats fed these diets,
which must be continuous to have a long – term influ-
ence on serum ferritin, total iron binding capacity, trans-
ferrin saturation, hemoglobin, haematocrit, red blood cell
and mean corpuscular volume.
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