Effect of Calcium and Phosphorus on Nonhaeme Iron Absorption and Haematogenic Characteristics in Rats

doi:10.4236/fns.2010.11003

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Food and Nutrition Sciences, 2010, 1, 13-18

doi:10.4236/fns.2010.11003 Published Online July 2010 (http://www.SciRP.org/journal/fns)

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.

Email: [email protected]

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

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:

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 +

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

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 + 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

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

37.54a ± 2

29.61b ± 1.4

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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