effect of iron and zinc supplementation and its discontinuation on lipid profile in rats

Accepted Manuscript

Title: Effect of iron and zinc supplementation and itsdiscontinuation on lipid profile in rats

Author: Joanna Kaluza Dawid Madej

PII: S0946-672X(14)00058-3DOI: http://dx.doi.org/doi:10.1016/j.jtemb.2014.04.002Reference: JTEMB 25522

To appear in:

Received date: 27-1-2014Revised date: 11-3-2014Accepted date: 9-4-2014

Please cite this article as: Kaluza J, Madej D, Effect of iron and zinc supplementationand its discontinuation on lipid profile in rats, Journal of Trace Elements in Medicineand Biology (2014), http://dx.doi.org/10.1016/j.jtemb.2014.04.002

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Effect of iron and zinc supplementation and its discontinuation on lipid profile in rats

Joanna Kaluza*, Dawid Madej

Department of Human Nutrition, Warsaw University of Life Sciences – SGGW, Warsaw,

Poland

Short title: iron and zinc supplementation and lipid profile

*Corresponding author:

Department of Human Nutrition

Warsaw University of Life Sciences – SGGW

Nowoursynowska 159C str., 02-776 Warsaw, Poland

tel: (+48) 22 59 37 114, fax: (+48) 22 59 37 117

e-mail: [email protected]

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Abstract

The aim of this research was to investigate whether combined iron/zinc supplementation is

more beneficial than iron supplementation alone from the perspective of the lipid profile in

rats. The study was conducted on 6-week male Wistar rats in 3 stages: 1) 4-week adaptation

to the diets: C (AIN-93M) and D (mineral mix without iron); 2) 4-week supplementation: 10-

times more iron or iron and zinc compared to C; 3) 2-week post-supplementation period (the

same diets as in the first stage). The iron and zinc content in serum was measured using ASA.

Total cholesterol (TC), HDL cholesterol (HDL-C), non-HDL cholesterol (non-HDL-C) and

triglycerides (TG) were determined. After 4-week supplementation (stage II) and post-

supplementation (stage III) periods combined iron/zinc supplementation decreased HDL-C

and increased non-HDL-C concentrations in control rats, and in contrast to iron

supplementation alone TG concentration decreased. After stage II combined iron/zinc

supplementation did not result in increased non-HDL-C and TG concentrations in iron

deficient rats in contrast to iron supplementation alone. After stage III both iron and

simultaneous iron/zinc supplementation were the cause of TC increase which was the result of

the increase of non-HDL-C but not HDL-C concentration in iron deficient rats. In conclusion,

there were no beneficial effects of simultaneous iron and zinc supplementation on the lipid

profile of rats fed control and iron deficient diets. Combined iron and zinc supplementation

may contribute to lower HDL-C and higher non-HDL-C concentrations.

Keywords: iron; lipid profile; rats; supplementation; zinc.

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Introduction

Hypercholesterolemia is one of the most relevant health problems among developed

societies and developing countries. This condition concerns about 46% of men and 51% of

women above 55 years old US population [1]. The results of both animal [2-4] and human [5,

6] studies indicated that iron overload may cause increased total cholesterol (TC), HDL

cholesterol (HDL-C) and triglycerides (TG). Moreover, high iron doses promote the

formation of oxidative stress which is a cause of lipids peroxidation [7-16], proteins and DNA

damage [11, 17-19].

On the other hand according to the World Health Organization about 1.62 billion

people worldwide suffer from anemia and insufficient iron status [20]. While iron

supplementation improves attention, intelligence quotient and concentration among anemic

children and pre-menopausal women [21], it also improves exercise capacity [22, 23] and the

quality of life among patients with heart diseases [23].

In this situation it is necessary to look for solutions, i.e. replenishment of iron

deficiency with minimizing the risk of adverse effects of high doses and overload of this

element in the organism. There are indications that zinc administration simultaneous with iron

can be effective in complement of iron deficiency [24-30] and can reduce an oxidative

damage induced by iron [24-26, 31], while zinc deficiency can impair the lipid profile [32]

and promote lipid oxidation [33].

Therefore, the aim of this study was to investigate whether combined iron and zinc

supplementation was more beneficial than iron supplementation alone from the perspective of

the lipid profile in rats. Furthermore, the influence of discontinuation of this treatment on the

lipid profile was also determined.

Materials and Methods

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Study design, diets and animals

The study was approved by the Third Local Ethics Commission in Warsaw. A hundred

thirty-two certificate (A5438-01, NIH Certified) male Wistar rats with initial weight 294 ± 20

g were purchased in the Polish Academy of Sciences Medical Research Center (Warsaw,

Poland). The animals were housed individually in glass-propylene cages in a temperature (21-

22oC) and humidity (55-60%) controlled laboratory with a 12-hour light/dark cycle.

The study design is shown in Table 1. After 4-week adaptation to the diets (C –

control or D – iron deficient) during 4-week supplementation period rats were fed diets

supplemented with iron (CSFe, DSFe) or iron and zinc (CSFeZn, DSFeZn). After intervention

stage during 2-week post-supplementation period rats were fed the same diets as in the

adaptation stage. All diets were based on AIN-93M recommendations [34] with some

modifications with iron or iron and zinc contents in mineral mixtures. The mineral mixture

added to the C diet contained 6.06 g ferric citrate and 1.65 g zinc carbonate per kg. The

mineral mixture without iron was added to the D diet, while in supplemented diets the

amounts of iron (CSFe, DSFe) and iron and zinc (CSFeZn, DSFeZn) in mineral mixtures

were 10-times higher compared to the C diet. Iron in mineral mixtures was in ferric form -

iron (III) citrate. In contrast to human this form of iron in comparison to ferrous form is less

effectively absorbed by rats [35]. The content of iron and zinc in experimental diets

determined by atomic spectrometry absorption is shown in Table 2. Rats had access to

ultrapure water ad libitum and were pair-fed with the group consuming the least amount of

diet.

Both iron and combined iron and zinc supplementation did not affect the body weight

among groups of rats fed various type of C- and D-diets. The impact of applied

supplementation on iron and zinc serum concentrations were observed only after stage III,

data were published earlier [30]. The content of iron in serum in the rats fed during stage II

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DSFe diet was statistically significantly higher than in the rats fed DSFeZn diet, but did not

differ from D group. The zinc concentration in serum was significantly higher in the rats fed

during stage II CSFe and CSFeZn diets compared to those fed C diet.

Blood collection and lipid profile

At the end of each stage, after overnight starvation, rats were anesthetized with an

intraperitoneal injection of thiopental. Blood was collected by a heart puncture and

immediately transferred into tubes containing serum separator. After 45 min blood samples

incubation in room temperature serum was separated by centrifugation at 3000 x g for 10 min

at 4oC.

Concentration of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C)

and triglycerides (TG) were determined in serum samples using commercial available kits

(Hydrex Diagnostics, Warsaw, Poland). TC concentration was determined using colorimetric

method based on the enzymatic cleavage of the cholesterol ester by cholesterol esterase and

oxidase (CHOD-PAP). The same method was used to determined HDL-C after previous

precipitation of VLDL and LDL by the polyethylene glycol. TG were measured colorimetric

method based on the enzymatic hydrolysis by glycerol-phosphate-oxidase, peroxidase and

ethyl-sulfopropyl-toluidine (GPO-POD-ESPT). Due to the fact that the Friedewald formula

generally does not accurately estimate VLDL-C levels in rat models especially in

hypercholesterolemic animals, it cannot be used to calculate LDL-C. Therefore, we combined

VLDL-C and LDL-C levels and presented them as non-high-density lipoprotein cholesterol

(non-HDL-C). The concentration of non-HDL-C was calculated by subtracting HDL-C from

TC.

Statistical analysis

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The data were presented as mean values ± standard deviation (SD) and were analysed

using Statistica software version 10. Homogeneity of variance was analyzed using Levene’s

test. The main effects and interactions were analyzed using a three-way analysis of variance.

Comparisons between the groups were conducted using LSD post-hoc test. The results with p-

values ≤ 0.05 were considered as statistically significant.

Results

Body weight, iron and zinc serum concentration

After supplementation (stage II) and post-supplementation (stage III) periods, both

iron and combined iron and zinc supplementation did not affect the body weight among

groups of rats fed various type of C- and D-diets. Data about the impact of applied

supplementation on iron and zinc serum concentrations were published earlier [30]. After

stage II the content of iron and zinc in serum did not differ between groups. The iron

concentration in serum in C, CSFe and CSFeZn rats was 2.01 ± 0.42, 2.26 ± 0.22 and 2.01 ±

0.60 µg/ml, respectively, while in D, DFe and DSFeZn rats was 2.13 ± 0.54, 2.66 ± 0.65 and

2.02 ± 0.37 µg/ml, respectively. After stage III the content of iron in serum in the rats fed

DSFe diet was statistically significantly higher than in the rats fed DSFeZn diet (2.33 ± 0.56

vs. 1.69 ± 0.43 µg/ml), but did not differ from D group (1.98 ± 0.32 µg/ml). After stage II the

content of zinc in serum C, CSFe and CSFeZn rats was 1.89 ± 0.54, 2.27 ± 0.37 and 1.64 ±

0.69 µg/ml, respectively, and in D, DFe and DSFeZn rats was 1.73 ± 0.38, 2.23 ± 0.41 and

2.00 ± 0.50 µg/ml, respectively. After stage III the zinc concentration in serum was

significantly higher in the rats fed CSFe and CSFeZn diets (2.09 ± 0.04 and 1.87 ± 0.26

µg/ml, respectively) compared to those fed C diet (1.32 ± 0.56 µg/ml).

Effect of iron and zinc supplementation on lipid profile

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Based on analysis of variance the statistically significant influence of the type of diet,

the type of supplementation and the stage of the experiment as well as interactions between

the diet and the supplementation were observed for all lipid profile parameters. Moreover,

significant interactions between the diet, the supplementation and the stage of the experiment

in TC, the supplementation and the stage of the experiment in non-HDL-C and the diet and

the stage of the experiment in TG concentration were found.

After 4-week adaptation period (stage I), concentration of TC, HDL-C, non-HDL-C as

well as TG did not differ statistically significantly between C and D rats (data not shown).

The effect of iron and zinc supplementation on lipid profile parameters was shown in Fig. 1.

After intervention (stage II) and post-intervention (stage III) periods, the rats fed CSFeZn diet

had significantly lower TC and HDL-C concentrations compared to the rats fed C and CSFe

diets, while TG concentration was significantly higher in CSFe group compared to C and

CSFeZn groups. Only after stage III in rats fed C-type of diets the impact of applied

intervention on non-HDL-C concentration was observed. Non-HDL-C concentration in the

rats fed CSFe and CSFeZn diets was significantly higher than in those fed C diet.

In the rats fed D-type of diets the differences after stages II and III in TC and non-

HDL-C concentration and after stage II in TG concentration were observed, while there was

no impact of applied intervention on HDL-C concentration. Directly after the intervention

period (stage II) TC was lower in DSFe and DSFeZn rats compared to D rats and non-HDL-C

was lower in DSFe rats compared to D and DSFeZn rats. After a two-week post-

supplementation period (stage III) in comparison to stage II TC concentration increased

significantly in rats fed DSFe and DSFeZn diets, and non-HDL-C concentration decreased in

D and DSFeZn rats. After stage III, TC and non-HDL-C were significant higher in DSFe and

DSFeZn rats in comparison to D rats. Furthermore, after stage II DSFe rats had a significantly

lower TG concentration than DSFeZn rats.

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Moreover, after stage II and stage III the significant impact of diet type on the

concentration of lipid profile parameters was found. After stage II, in comparison to the rats

fed D-type diets those fed C diet had higher HDL-C and lower non-HDL-C concentrations,

those fed CSFe diet had higher TC, non-HDL-C and TG, while CSFeZn rats had lower non-

HDL-C as well as TG concentrations. After stage III, C group of rats had higher TC and

HDL-C concentrations, while CSFe group had higher TG concentration compared to

corresponding D-type groups.

Discussion and conclusion

Our data suggested that iron supplementation alone had an adverse effect on lipid

parameters in rats fed control or iron deficient diets. In control rats non-HDL-C (after stage

III) and TG (after stages II and III) increased, but not TC and HDL-C concentrations, while in

iron deficient rats TC and non-HDL-C were affected by decreasing these parameters after

stage II and increasing after stage III. The results of other animal and human studies on effect

of iron supplementation on lipid metabolism are incoherent. Our results confirm some of

them, indicated that high iron content in rats diet was associated with high blood

concentration of TC [2, 4, 36], TG [2, 4], LDL-C as well as VLDL-C [2], but not HDL-C [2].

Among anemic girls (hemoglobin, Hb < 8.0 g/dL), 14-19 years, TC and TG, but not HDL-C

and LDL-C, were significantly lower than in non-anemic (Hb ≥ 14.0 g/dL), moreover, iron

supplementation of anemic girls was the cause of a significant increase of TC and TG in their

serum [5]. In contrast, in other studies iron overload in control and hypercholesterolemic rats

was connected with a significant reduction of TC and redistribution of cholesterol among the

various lipoprotein fractions, with an increase in HDL-C and a decrease in LDL-C [37], and

in another study with a significant increase TG, but not TC concentration [38]. Moreover, it is

known that iron overload is a cause of lipid peroxidation in both animals [7, 9, 13, 15] and

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humans [8, 10, 12, 14, 16], while zinc supplementation protects lipids against oxidation

changes [33, 39].

Generally, in our study we did not observe beneficial effects of simultaneous iron and

zinc supplementation in rats fed control and iron deficient diets on lipid profile parameters. In

control rats combined iron and zinc supplementation was related to lower TC (after stages II

and III), but it was a result of significantly lower HDL-C (after stages II and III) and higher

non-HDL-C (after stage III) concentrations. In iron deficient rats the results of combined iron

and zinc supplementation on TC depended on the stage of the study, in comparison with non-

supplemented rats TC decreased significantly after stage II and next increased significantly

after stage III. Moreover, after stage II the simultaneous iron and zinc supplemented rats had a

significantly higher TG compared to compared to the rats fed iron supplemented diet and after

stage III had higher non-HDL-C concentration compared to non-supplemented rats.

Only a few earlier animal studies have examined the effect of combined iron and zinc

supplementation on blood lipid profile parameters [2, 36, 40]. None of the mentioned study

showed beneficial effects of simultaneous iron and zinc supplementation on TC [2, 36, 40]

and TG [2, 40], what is more, in one study [40] after combined iron and zinc supplementation

LDL-C significantly increased and HDL-C concentrations decreased. However, there are

indications which suggested that insufficient zinc content in rats diet was related to an

increase of lipid peroxidation [41], TC, TG, LDL-C concentrations and a decrease of HDL-C

[32]. However, other researchers did not support finding presented above and did not show

significant interactions between diet zinc amounts and TC [40, 42], TG and HDL-C [40].

Also, the results of a meta-analysis of thirty three randomized controlled trials indicated the

lack of effect of zinc supplementation on TC, LDL-C, HDL-C and TG concentrations in

subject with various health status, while in individuals classified as healthy it was associated

with a significant decrease in HDL-C concentration [43].

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Although, after stages II and III the iron serum concentration in D rats did not differ

significantly from C rats, the iron level in the liver in D rats was depleted, and was 2.8-fold

and 3.2-fold lower after stages II and III respectively than in C rats [31]. After stages II and

III, serum ferritin and hemoglobin concentration in D rats was significantly lower (p-value <

0.05) than in C rats. After stage II, in the rats DSFe and DSFeZn diet ferritin and hemoglobin

concentration increased significant in comparison to the rats fed constantly D diet [44]. The

positive effect of used dietary intervention on iron status had the prolonged effect and was

visible two weeks after supplementation discontinuation.

Whilst the results of other investigations are limited only to intervention period, our

studies examined the prolonged effect of used intervention and reflected a situation often

encountered among humans when no diet corrections were made after the termination of

supplementation. We observed the prolonged effect of both iron and iron and zinc

supplementation on analyzed parameters of lipids metabolism in rats fed C- and/or D-type of

diets. After stage III in rats fed C-type of diets for TC, HDL-C and TG the findings had the

same direction as after stage II, while after stage III compared to stage II in rats fed D-type of

diets we observed an opposite effect of applied intervention for TC and non-HDL-C in

comparison to non-supplemented rats. Moreover, the effect of applied intervention on non-

HDL-C concentration in rats fed C-type of diets was delayed and visible only after the post-

intervention period (stage III). These observations indicate a need for a post-intervention

observation in such studies.

In summary, we did not observe beneficial effects of simultaneous iron and zinc

supplementation on lipid profile parameters in rats fed control and iron deficient diets. The

results indicated that combined iron and zinc supplementation may contribute to lower HDL-

C and higher non-HDL-C concentrations. Due to the fact that both iron and combined iron

and zinc supplementation can impair lipid metabolism and thus may contribute to

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cardiovascular disease, further studies on minimizing the risk of adverse effects of iron

supplementation are necessary.

Acknowledgments

The study was supported by a grant from the Ministry of Science and Higher

Education (MNiSZW), Poland (No. N N312 329735).

Conflict of Interest

On behalf of all authors the corresponding author states that there is no conflict of

interest.

Acknowledgments

The study was supported by a grant from the Ministry of Science and Higher

Education (MNiSZW), Poland (No. N N312 329735). We thank the head of Department of

Human Nutrition WULS - SGGW – Prof. Anna Brzozowska for general support.

Authors contributions

JK: study concept and design, JK and DM: oversee the animal and diet manipulations,

JK and DM: conduct the animal experiments, JK: perform analytical determinations of study

parameters, JK: analysis and interpretation of data, JK: perform the statistical analysis and

draft the manuscript, JK: critical revision of the manuscript.

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Table 1 Experimental design

Diet/Group

Stage I

adaptation to diets

(4 weeks)

Stage II

supplementation period

(4 weeks)

Stage III

post-supplementation

period

(2 weeks)

C control diet

CSFe supplemented with Fe

CSFeZn

control diet

supplemented with Fe and Zn

control diet

D Fe-deficient diet

DSFe supplemented with Fe

DSFeZn

Fe-deficient diet

supplemented with Fe and Zn

Fe-deficient diet

C – control diet; D – iron deficient diet; CSFe, DSFe – diets supplemented with iron;

CSFeZn, DSFeZn – diets supplemented with iron and zinc. Number of animals is 6-7 in each

group.

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Table 2 Content of iron and zinc in experimental diets determined by atomic spectrometry

absorption

Element

(mg/kg diet) C CSFe CSFeZn D DSFe DSFeZn

Fe 48.4 470 490 7.4 470 490

Zn 42.6 44.7 412 43.4 44.7 412

C – control diet; D – iron deficient diet; CSFe, DSFe – diets supplemented with iron;

CSFeZn, DSFeZn – diets supplemented with iron and zinc.

The content of other diet ingredients was based on AIN-93M recommendations [34].

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0.0

0.5

1.0

1.5

2.0

2.5

C CSFe CSFeZn D DSFe DSFeZn C CSFe CSFeZn D DSFe DSFeZn

Tota

l ch

ole

ster

ol (

mm

ol/

l)

a

b

a a

b b

*

#

a

b

a

*

a

b b

#

0.0

0.5

1.0

1.5

2.0

2.5


HD

L-C

(m

mo

l/l)

b

a a

b

#

a

*

a

*

0.0

0.1

0.2

0.3

0.4

0.5

0.6


no

n-H

DL-

C (

mm

ol/

l)

a

b

b b

*

#

a

a

*

a

b b

*

#

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4


Trig

lyce

rid

es

(mm

ol/

l)

a

b

ab

a

b

*

#

a

b

a

*

a

*

STAGE II STAGE III STAGE II STAGE III Figure

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Fig. 1. Effect of iron and zinc supplementation on total cholesterol, HDL-C, non-HDL-C, and triglycerides after supplementation (stage II) and

post-supplementation (stage III) periods in blood of rats fed experimental diets.

C–control diet; D–iron deficient diet; CSFe, DSFe– diets supplemented with iron; CSFeZn, DSFeZn– diets supplemented with iron and zinc.

a,b – different letters indicate statistically significant differences within a stage of experiment for the same type of diet, p-value ≤ 0.05 (LSD

test); * – a statistically significant difference between a group of rats fed C-type diets compared to a corresponding group of rats fed D-type diets,

p-value ≤ 0.05; # - a statistically significant difference between stage II and stage III, p-value ≤ 0.05 (LSD test); Bars represented mean ± SD of

6-7 animals/group.

effect of iron and zinc supplementation and its discontinuation on lipid profile in rats

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