Eur J Nutr DOI 10.1007/s00394-013-0610-y

ORIGINAL CONTRIBUTION

The effect of iron and zinc supplementation and its discontinuation on liver antioxidant status in rats fed deficient diets Joanna Kaluza • Dawid Madej • Anna Rusaczonek Ewa Siedlecka • Barbara Pietruszka



Received: 28 March 2013 / Accepted: 20 October 2013  Springer-Verlag Berlin Heidelberg 2013

Abstract Purpose The aim was to investigate the effect of iron or combined iron/zinc supplementation on rat liver antioxidant status. Methods The 6-week male Wistar rats were examined in 3 stages: (1) 4-week adaptation to the diets (C—control AIN-93M diet, D—iron deficient and R—with 50 % reduction in all vitamin and mineral amounts); (2) 4-week supplementation with the same regimen enriched with tenfold more iron or iron/zinc; (3) 2-week post-supplementation period (the same diets as in the stage I). Results Combined iron/zinc supplementation similarly to iron supplementation alone significantly (p values B 0.05) increased the iron content in the liver in D and R rats after stages II and III. Moreover, iron/zinc supplementation compared to iron supplementation alone significantly decreased the liver concentration of 8-isoprostane (after stage II in D and after stage III in R rats), protein carbonyl groups (only after stage III in R rats) and 8-hydroxy-2deoxyguanosine (after stage II in R and after stage III in D and R rats). In rats fed R-type of diets after stage II hepatic superoxide dismutase (SOD) and catalase (CAT) activity, but not glutathione peroxidation activity and total antioxidant capacity, was lower in iron and iron/zinc supplemented than in non-supplemented rats, whereas after stage III in iron/zinc supplemented SOD was lower and CAT J. Kaluza (&)  D. Madej  B. Pietruszka Department of Human Nutrition, Warsaw University of Life Sciences, SGGW, Nowoursynowska 159C Str., 02-776 Warsaw, Poland e-mail: [email protected] A. Rusaczonek  E. Siedlecka Department of Plant Genetics, Breeding and Biotechnology, Warsaw University of Life Sciences, SGGW, Warsaw, Poland

activity was higher in comparison with non-supplemented and iron supplemented rats. Conclusions The simultaneous iron/zinc supplementation can protect liver against peroxidative damage induced by high doses of iron during and after the intervention in rats fed iron-deficient diet and diet with reduced amounts of vitamins and minerals. The post-intervention observation is relevant because the effect may be delayed and visible only after this period. Keywords Iron  Zinc  Liver  Antioxidant status  Rats  Supplementation

Introduction It is known that the prevalence of anaemia and iron deficiency is common among some groups of people such as infants, neonatal and preschool children, among women at reproductive age as well as pregnant and breastfeeding women and people with absorption disorders [1]. Generally, iron-deficient population suffers also from zinc deficiency. This is due to the fact that the same group of products, namely meat and meat products, is the main source of well-absorbed iron and zinc and the same dietary factors can interfere (e.g. phytates and tannins) both iron and zinc metabolism [2, 3]. Usually iron supplementation is recommended to people without assessment of zinc status and without zinc supplementation, which can impair balance between these minerals in the diet. It is generally considered that iron has pro-oxidative properties by catalyses the conversion of superoxide and hydrogen peroxide into hydroxyl radicals in the Fenton reaction [4]. High doses of it may lead to oxidative damage of body cells, particularly liver cells, where this element is

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Eur J Nutr

Materials and methods

were bought from the Medical Research Centre of Polish Academy of Sciences (Warsaw, Poland), and they were housed in plexiglass cages in the laboratory at 21–22 C, 55–60 % humidity and with a 12-h light/dark cycle. The studies were approved by the Third Local Ethics Commission in Warsaw and have been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. The studies’ design is presented in Fig. 1. Both studies were divided into 3 stages: (1) 4-week adaptation to a diet (C—control, D—iron-deficient or R—diet with reduced amounts of all vitamins and minerals), (2) 4-week supplementation period (10 times more iron: CSFe, DSFe, RSFe or iron and zinc: CSFeZn, DSFeZn, RSFeZn were added to the diets than to the control diet) and (3) 2-week post-supplementation period (the same diets as during the adaptation stage). During the experiments, rats had access to water ad libitum and were pair-fed with the group consuming the least amount of diet. All diets were prepared according to AIN-93M recommendations [13] with some modifications in added mineral and vitamin mixtures. Mineral mix used to prepare D diet did not contain iron. In R diets, added amount of vitamin and mineral mixtures was reduced by 50 % compared to C diet. Composition of the experimental diets is presented in Table 1.

Animal study, study design and diets

Tissue collections and homogenization

Two studies were conducted on male Wistar rats with a mean initial weight 294 ± 20 g in study I and 296 ± 23 g in study II. The certificate rats (A5438-01, NIH Certified)

At the end of each stage, after an overnight fast, the rats were anesthetized with an intraperitoneal injection of thiopental. Blood was taken by heart puncture, and then the

accumulated [4] and may be the cause of lipid peroxidation [5–7] as well as protein and DNA damage [7, 8]. Whereas, zinc as indirect antioxidant can protect the body cells against the damage induced by free radicals including excess of iron [4]. Animal studies indicated that combined iron and zinc supplementation reduced oxidative damage caused by high doses of iron [9, 10] and such a practise is effective in correction of iron deficiency [9–12]. The path towards the understanding of the iron supplementation with minimizing the risk of the adverse effect of high doses and excess of this element in the organism is very relevant. To the best of our knowledge, no study has examined whether the simultaneous zinc and iron supplementation can protect liver cells against oxidative damage caused by high doses of iron supplements and after discontinuation of this treatment. Therefore, the aim of this study was to investigate the effect of combined iron and zinc supplementation on liver antioxidant status of rats fed iron-deficient diet or diet with reduced amounts of all vitamins and minerals. Furthermore, the influence of discontinuation of applied intervention on antioxidant status in liver was determined.

Stage I adaptation to diets

Stage II supplementation period

Stage III post-supplementation period

(4 weeks)

(4 weeks)

(2 weeks)

Control diet (C) Control diet (C)

Supplemented with Fe (CSFe)

Control diet (C)

Supplemented with Fe and Zn (CSFeZn)

STUDY I Fe deficient diet (D) Fe deficient diet (D)

Supplemented with Fe (DSFe)

Fe deficient diet (D)

Supplemented with Fe and Zn (DSFeZn)

Diet with reduced quantity of vitamins and minerals (R)

STUDY II

Diet with reduced quantity of vitamins and minerals (R)

Supplemented with Fe (RSFe) Supplemented with Fe and Zn (RSFeZn)

Fig. 1 Design of the studies

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Diet with reduced quantity of vitamins and minerals (R)

Eur J Nutr Table 1 Content of iron and zinc in experimental diets Elementb (mg/kg diet)

Dieta C

CSFe

CSFeZn

Fe

48.4

470

490

7.4

Zn

42.6

412

43.4

44.7

D

DSFe

DSFeZn

R

RSFe

RFeZn

470

490

32.3

501

542

412

28.2

29

500

44.7

C control diet, D iron-deficient diet, R diet with reduced amounts of vitamin and mineral mixtures; CSFe, DSFe, RSFe diets supplemented with iron, CSFeZn, DSFeZn, RSFeZn diets supplemented with iron and zinc a

Moreover, diets included (per kg diet): wheat starch (C, D diets 621 g; R diets 643 g), casein 140 g, sucrose 100 g, soybean oil 40 g, cellulose 50 g, modified mineral mix AIN-93M (C, D diets 35 g; R diets 17,5 g), vitamin mix AIN-93-VX (MP Biomedicals, LLC, No. 960402; C, D diets 10 g; R diets 5 g), L-cystine 1.8 g, choline bitartrate 2.5 g, and t-butylhydroquinone 0.008 g

b

The contents of Fe and Zn in diets determined by FAAS

livers were removed, rinsed with an ice-cold 0.9 % NaCl solution (Merck, Darmstadt, Germany), dried on filter paper, weighted and stored at -80 C until the analysis. Before all analyses, except iron and zinc determination, liver samples were homogenized in 50 mM phosphate buffer (pH 7.0–7.4) containing 1 mM EDTA (Sigma-Aldrich, St. Louis, MO, USA) using a motor-driven Bio-Gen Pro200 homogenizer (ProScientific, Oxford, CT, USA). To determine total antioxidant capacity (TAC), tissues were homogenized with buffer containing 0.9 % NaCl without EDTA. In addition, the buffer used to homogenization samples to determine catalase (CAT) activity contained 0.1 % bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) and to determine 8-isoprostane contained 0.005 % butylhydroxytoluene (BHT) (Sigma-Aldrich, St. Louis, MO, USA). The homogenates were centrifuged at 10,0009g for 10 min at 4 C. Iron and zinc determination Approximately 0.5–1.0 g wet mass of liver samples and *1.0 g dry mass of experimental diets were digested with 65 % HNO3 (Merck, Darmstadt, Germany) for 10 min at 210 C using microwave digestion system (Mars5, CEM, Matthews, USA). Iron and zinc content was determined by flame atomic absorption spectrometry (FAAS) using a spectrophotometer Solaar 989 (Unicam Atomic Absorption, Cambridge, UK) as described previously [14]. Iron and zinc standard curves were prepared by diluting iron and zinc standard reference materials (Merck, Darmstadt, Germany) in a range from 0 to 5.0 lg/cm3. Antioxidant status TAC was measured using trolox equivalent antioxidant capacity (TEAC) assay based on the capacity of a sample to inhibit the ABTS?• [radial monocation of 2,20 -azinobis(3-ethylbenzothiazoline-6-sulphonic acid)] compared with a reference antioxidant standard—trolox (a water soluble

vitamin E analogue) (Sigma-Aldrich, St. Louis, MO, USA) [15]. Total superoxide dismutase (SOD) activity was measured in samples incubated with xanthine oxidase and 2-(4indophenyl)-5-phenyltetrasodium chloride (INT) at 505 nm and 37 C using ready-to-use RANSOD kit (Randox Laboratories Ltd., Ardmore, UK). Glutathione peroxidase (GPx) activity was determined in samples incubated with cumene hydroperoxide at 340 nm and 37 C using RANSEL kit (Randox Laboratories Ltd., Ardmore, UK). The determination of peroxidatic CAT activity based on the reaction of the enzyme with methanol (POCH, Gliwice, Poland) in the presence of H2O2 (Sigma-Aldrich, St. Louis, MO, USA) [16]. The formaldehyde produced was determined with purpald (4-amino-3-hydrazino-5-mercapto1,2,4-triazole measured) (Sigma-Aldrich, St. Louis, MO, USA) as a chromogen, and the intensity of the formed coloured complex was measured spectrophotometrically at 540 nm. Lipids, protein and DNA damage The lipids peroxidation level in liver samples was determined using the specific enzyme immunoassay (EIA) kit based on 8-isoprostane measurements (Cayman Chemical Company, Michigan, USA). The protein oxidation damage was identified by measuring protein carbonyl groups content in samples using the commercial assay kit (Cayman Chemical Company, Michigan, USA). The oxidative DNA damage was assessed by an 8-hydroxy-2-deoxyguanosine (8-OHdG) enzyme-linked immunosorbent assay (Cayman Chemical Company, Michigan, USA). DNA was isolated from liver tissue using the DNA extraction kit (A&A Biotechnology, Gdynia, Poland) and digested with nuclease P1 (Sigma-Aldrich, St. Louis, MO, USA). The level of DNA in samples was determined using spectrophotometer NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA).

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Eur J Nutr Table 2 Effect of iron or iron and zinc supplementation on liver mass and level of iron and zinc in liver after supplementation (stage II) and post-supplementation (stage III) periods (mean ± SD) Diet/group

Liver mass (g w.m.)

Iron (lg/g w.m.)

Zinc (lg/g w.m.)

Stage I

Stage I

Stage I

Stage I

Stage II

Stage III

Stage II

Stage III

Stage II

Stage III

Stage II

Stage III

Study I C

128 ± 16à

11.4 ± 1.5

C

C

10.8 ± 0.7

10.9 ± 1.7

159 ± 25a,

CSFe

C

11.3 ± 1.6

10.8 ± 1.6

CSFeZn D

C

10.4 ± 0.6 11.2 ± 1.09

D

D

10.7 ± 0.8

34.0 ± 1.9 #

175 ± 37#

35.6 ± 0.9

36.9 ± 2.4

170 ± 25ab

177 ± 51

35.9 ± 1.6

36.5 ± 2.6

10.8 ± 1.2

187 ± 33b,# 56 ± 15

165 ± 30

37.3 ± 2.9 35.3 ± 2.1

36.2 ± 1.3

10.2 ± 1.5

56 ± 13a

54 ± 8a b

35.0 ± 3.0

36.4 ± 2.3

DSFe

D

11.0 ± 1.2

10.9 ± 2.0

149 ± 30

138 ± 21b

35.4 ± 2.0

34.8 ± 2.1

DSFeZn

D

11.1 ± 1.1

10.6 ± 1.2

152 ± 25b

151 ± 35b

35.8 ± 1.4

35.1 ± 3.5

Study II 9.4 ± 0.6 

R R

R

143 ± 18à

12.3 ± 1.1

a

38.6 ± 2.5

12.4 ± 1.2

136 ± 24a

120 ± 11a

b

11.5 ± 1.5

b

161 ± 10

159 ± 25

b

11.2 ± 1.2

166 ± 21b

150 ± 31b

RSFe

R

11.2 ± 0.8

RSFeZn

R

10.4 ± 1.0b

33.3 ± 3.5a

36.2 ± 2.8

35.9 ± 2.5ab

36.2 ± 4.0

38.0 ± 2.5b

37.1 ± 2.4

Variance analysis, p value (Study I) Diet (C vs. D)

NS

\0.001

0.05

Suppl (non-suppl vs. Fe vs. FeZn)

NS

\0.001

NS

Stage (II vs. III)

NS

NS

NS

Diet 9 suppl

NS

\0.001

NS

Diet 9 stage Suppl 9 stage

NS NS

NS NS

NS NS

Diet 9 suppl 9 stage

NS

NS

NS

Suppl (non-suppl vs. Fe vs. FeZn)

0.012

\0.001

0.017

Stage (II vs. III)

NS

NS

NS

Suppl 9 stage

NS

NS

NS

Variance analysis, p value (Study II)

Different letters indicate statistically significant differences between groups of rats fed the same type of diet within a stage of experiment, p value B 0.05 (LSD test) w.m. wet mass, C control diet, D iron-deficient diet, R diet with reduced amounts of vitamin and mineral mixtures, CSFe, DSFe, RSFe diets supplemented with iron, CSFeZn, DSFeZn, RSFeZn diets supplemented with iron and zinc NS not significant, p value [ 0.05; number of animals is 6–7 in each group #

A statistically significant difference between a group of rats fed C-type of diets compared to a corresponding group of rats fed D-type of diets, p value B 0.05 (LSD test)

 

Statistically significant differences between stage I and stage II and between stage I and stage III in rats fed a non-supplemented diet through three stages, p value B 0.05 (LSD test) à A statistically significant difference between stage I and stage III in rats fed a non-supplemented diet through three stages, p value B 0.05 (LSD test)

Statistical analysis The data were presented as mean values ± standard deviation (SD) and were analysed using Statistica software version 10.0. Homogeneity of variance was analysed using Levene’s test. Due to the fact that our experiment was

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based on two separately conducted studies the statistical analyses were performed individually for each study. Comparisons between groups were conducted using a three-way (study I) and two-way (study II) analysis of variance and LSD post hoc test. The results with p values B 0.05 were considered as statistically significant.

Eur J Nutr

Results Liver weight, iron and zinc liver concentration In study I, the liver weight did not significantly differ in the rats fed C or D diet constantly from stage I to stage III (Table 2). Moreover, there were no differences in the liver weight between groups of rats after supplementation (stage II) and post-supplementation (stage III) periods. In study II in the rats fed R diet through three stages, the liver weight was significantly higher after stages II and III in comparison with stage I, and after stage II, RSFe and RSFeZn rats had a statistically lower weight of livers compared to R rats. In study I, a significant interaction between diet and supplementation was found. A regular significant increase in iron content in the liver was observed in C rats, but not in D, from stage I to stage III (Table 2). After a 4-week supplementation period (stage II), the rats fed DSFe and DSFeZn diets had a significantly higher iron level in the liver than the rats fed D diet. In the rats fed C-type of diets, only CSFeZn group had a significantly higher level of iron in comparison with C group. After the post-supplementation period (stage III), the effect of applied supplementation in D rats, but not in C, was still observed. After stage II, the iron content in the liver in C and CSFeZn rats and after stage III in C rats was significantly higher compared to corresponding D-type of diets. Opposite to C rats in the rats fed R diet through three stages, a regular decrease in iron level in the liver was found. Moreover, the impact of used intervention in the rats fed R-type diets was observed. After stages II and III, the rats fed RSFe and RSFeZn diets during stage II had a significantly higher level of iron in the liver than those fed R diet. The significant influence of supplementation on zinc content in the liver was shown only in study II. The level of zinc in the liver of RSFeZn rats was significantly higher in comparison with R rats, but RSFe group did not differ from R and RSFeZn groups. Effect of iron and zinc supplementation on TAC and antioxidant enzymes activity In study I in the rats fed non-supplemented diets (C or D) from stage I to stage III, we did not observe any statistically significant changes of TAC and activity of antioxidant enzymes in the liver (Table 3). What is more, after stages II and III regardless the diet (C or D), there was no effect of iron or iron and zinc supplementation on TAC and antioxidant enzymes activity, while the significant differences in CAT activity in the liver depending on the stage of the experiment were found. After stage III, the rats fed

during stage II D and DSFe diets had higher CAT activity than after stage II. In study II, a regular significant increase in TAC and a decrease in GPx activity were shown in the rats fed R diet constantly from stage I to stage III (Table 3). For CAT activity, a significant interaction between supplementation and a stage of experiment was found and the impact of applied supplementation on SOD activity was observed. After stage II, RSFe and RSFeZn rats had lower SOD and CAT activity in the liver compared with R rats. After stage III in RSFeZn rats’ livers, SOD activity was significantly lower, but CAT activity was significantly higher than in R and RSFe rats. Moreover, after stage III compared with stage II, SOD activity increased significantly in the rats fed during stage II RSFe diet, while CAT activity decreased in the rats fed during stage II R diet and increased in the rats fed RSFeZn diet. Effect of iron and zinc supplementation on lipids, protein and DNA damage In the livers of the rats fed non-supplemented diets (C, D or R) through three stages, a regular increase in concentration of 8-isoprostane in C rats, 8-OHdG in C and D rats and protein carbonyl groups in R rats was observed (Fig. 2). In study I, significant interactions between supplementation and a stage of experiment and between supplementation, a stage of experiment and diet in 8-isoprostane levels were found. After the supplementation period (stage II), 8-isoprostane level in the livers of the rats fed CSFe and CSFeZn diets was significantly higher than in those fed C diet, and in DSFe rats was significantly higher than in D and DSFeZn rats (Fig. 2A). After the post-supplementation period (stage III), the rats fed in stage II CSFeZn diet had a lower 8-isoprostane level in the liver compared with those fed C diet. Moreover, the level of 8-isoprostane in the liver in the rats fed CSFeZn diet during stage II was significantly reduced after discontinuation of supplementation. After stage II CSFeZn group and after stage III C group had significantly higher 8-isoprostane concentration than DSFeZn and D groups, respectively. Similarly to study I also in study II, a significant interaction between supplementation and a stage of experiment in 8-isoprostane in the liver was observed. After stage III, the rats fed in stage II RSFe diet had a significantly higher level of this parameter than R and RSFeZn rats. Moreover, 8-isoprostane concentration increased significantly in RSFe rats after the post-supplementation stage in comparison with stage II. In study I and study II directly after stage II, the effect of dietary intervention on concentration of protein carbonyl groups in the liver was not observed (Fig. 2B). However, after stage III, the rats fed in stage II CSFe diet had a

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Eur J Nutr Table 3 Effect of iron or iron and zinc supplementation on total antioxidant capacity and liver antioxidant enzymes activity after supplementation (stage II) and post-supplementation (stage III) periods (mean ± SD) Diet/group

TAC (lmol TE/g w.m.)

SOD (U/mg protein)

GPx (U/mg protein)

CAT (U/mg protein)

Stage I

Stage I

Stage I

Stage I

Stage I

Stage II

Stage III

Stage II

Stage III

Stage II

Stage III

Stage II

C

C

CSFe CSFeZn

Stage III

Stage II

39.8 ± 2.8

39.8 ± 1.2

19.6 ± 3.8

20.8 ± 6.2

C

38.2 ± 2.6

38.5 ± 3.8

20.0 ± 5.5

C

38.8 ± 3.8

40.7 ± 3.0

19.3 ± 7.6

D

D

40.7 ± 1.5

41.4 ± 1.6

DSFe

D

39.4 ± 2.2

DSFeZn

D

40.5 ± 1.5

Stage III

0.54 ± 0.05

0.57 ± 0.12

0.57 ± 0.07

0.64 ± 0.14

20.9 ± 6.8

0.51 ± 0.07

0.50 ± 0.12

0.52 ± 0.12

0.58 ± 0.12

20.8 ± 5.2

0.62 ± 0.09

0.60 ± 0.17

0.45 ± 0.10

0.53 ± 0.10

15.1 ± 4.7

26.3 ± 5.5

0.70 ± 0.18

0.72 ± 0.18

0.49 ± 0.08*

40.4 ± 2.2

19.4 ± 2.2

20.7 ± 6.4

0.53 ± 0.14

0.69 ± 0.18

0.54 ± 0.05*

0.61 ± 0.04

38.5 ± 3.1

19.0 ± 5.8

17.2 ± 2.7

0.53 ± 0.19

0.68 ± 0.14

0.54 ± 0.06

0.56 ± 0.09

0.54 ± 0.10

0.70 ± 0.06a*

Study I C

39.8 ± 1.3

D

19.8 ± 4.8

40.8 ± 2.8

0.66 ± 0.13

20.8 ± 8.0

0.52 ± 0.08

0.56 ± 0.16

0.54 ± 0.15 0.65 ± 0.10

Study II 40.3 ± 1.1à

R R

R

RSFe

R

41.8 ± 1.6 41.4 ± 1.4

RSFeZn R 41.0 ± 1.1 Variance analysis, p value (Study I)

0.71 ± 0.16à

32.6 ± 6.4 42.9 ± 1.0 42.6 ± 1.6 42.9 ± 1.8

27.0 ± 5.0

a

19.5 ± 5.5

b,*

18.0 ± 3.8

b

34.7 ± 6.4

a

32.3 ± 4.8

a

22.6 ± 4.8

b

0.67 ± 0.10 0.71 ± 0.17 0.62 ± 0.08

0.47 ± 0.10 0.65 ± 0.09 0.63 ± 0.17

0.48 ± 0.06a

b*

0.60 ± 0.05b

0.52 ± 0.12 0.50 ± 0.05

Diet (C vs. D)

NS

NS

0.043

NS

Suppl (non-suppl vs. Fe vs. FeZn)

NS

NS

NS

NS

Stage (II vs. III)

NS

NS

NS

0.002

Diet 9 suppl

NS

NS

NS

NS

Diet 9 stage

NS

NS

NS

NS

Suppl 9 stage

NS

NS

NS

NS

Diet 9 suppl 9 stage

NS

NS

NS

NS

NS

0.016

NS

NS

Stage (II vs. III)

0.024

\0.001

NS

NS

Suppl 9 stage

NS

NS

NS

0.011

0.52 ± 0.10a

b

Variance analysis, p value (Study II) Suppl (non-suppl vs. Fe vs. FeZn)

Different letters indicate statistically significant differences between groups of rats fed the same type of diet within a stage of experiment, p value B 0.05 (LSD test) TAC total antioxidant capacity, SOD superoxide dismutase, GPx glutathione peroxidase, CAT catalase C control diet, D iron-deficient diet, R diet with reduced amounts of vitamin and mineral mixtures, CSFe, DSFe, RSFe diets supplemented with iron, CSFeZn, DSFeZn, RSFeZn diets supplemented with iron and zinc NS not significant, p value [ 0.05; Number of animals is 6–7 in each group *

A statistically significant difference compared to the corresponding group of rats after stage III, p value B 0.05

à

A statistically significant difference between stage I and stage III in rats fed a non-supplemented diet through three stages, p value B 0.05 (LSD test)

significantly higher level of protein carbonyl groups compared with the rats fed C diet. Also, the concentration of this parameter after stage II in CSFe rats and after stage III in C rats was significantly lower than in the corresponding D groups of rats. Moreover, in study II, a significant interaction between supplementation and a stage of experiment in the level of protein carbonyl groups was found. After stage III in the rats fed in stage II RSFeZn

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diet, the level of this parameter was significantly lower than in the rats fed RSFe diet. Furthermore, the level of carbonyl protein groups in the livers of the rats fed in stage II RSFeZn diet was significantly reduced after discontinuation of supplementation. The effect of applied supplementation on the level of DNA damage in the liver was observed after stage II in the rats fed R-type of diets and after stage III in the rats fed

Eur J Nutr

STUDY I

STUDY II

(A)

(B)

(C)

Fig. 2 Effect of iron and zinc supplementation on (A) 8-isoprostane, (B) protein carbonyl groups and (C) 8-OHdG level after supplementation (stage II) and post-supplementation (stage III) periods in liver of rat fed experimental diets. C control diet; D iron-deficient diet; R diet with reduced amounts of vitamin and mineral mixtures; CSFe, DSFe, RSFe diets supplemented with iron; CSFeZn, DSFeZn, RSFeZn diets supplemented with iron and zinc. a,b different letters indicate statistically significant differences between groups of rats fed the same type of diet within a stage of experiment, p value B 0.05 (LSD

test); *represents a statistically significant difference between stage II and stage III, p value B 0.05; #a statistically significant difference between a group of rats fed C-type of diets compared to a corresponding group of rats fed D-type of diets, p value B 0.05 (LSD test); àa statistically significant difference between stage I and stage III in rats fed a non-supplemented diet through three stages, p value B 0.05 (LSD test); Bars represented mean ± SD of 6–7 animals/group

D-type and R-type of diets (Fig. 2C). After stage II, 8-OHdG level was significantly higher in the liver of RSFe rats compared to R and RSFeZn rats, while after stage III, this parameter in the rats fed RSFeZn diet during the

supplementation period was significantly lower compared to those fed RSFe diet. Also, after stage III, 8-OHdG level was significantly lower in the rats fed in stage II DSFeZn diet compared with those fed D and DSFe diets.

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Eur J Nutr

Discussion The results of the presented studies indicate that simultaneous administration of zinc and iron can protect the liver against oxidative damages caused by excesses of iron in rats fed iron-deficient diet and diet with a reduced amount of all vitamins and minerals. Combined iron and zinc supplementation compared to iron supplementation alone decreased liver lipid peroxidation (after stage II in D rats and after stage III in R rats), protein oxidation (after stage III in R rats) and DNA damage (after stage II in R rats and after stage III in D and R rats). The animal studies on the effect of iron and zinc supplementation and discontinuation of this dietary intervention on liver antioxidant status are lacking. According to our knowledge, only in one previous animal study described in three papers [9, 10, 17] the effect of combined iron and zinc supplementation on intestine oxidative damage was examined. In the study mentioned above, rats fed diet supplemented with iron and zinc had a significantly lower concentration of thiobarbituric acid reactive substances (TBARS) and protein carbonyl groups in the intestine compared with rats fed diet supplemented with iron alone. Moreover, SOD, CAT and GPx activity in the intestine of rats supplemented with iron and zinc was lower than in rats supplemented with iron alone. In our studies, the impact of used dietary intervention on antioxidant enzymes activity was not as visible as in the study mentioned above and concerned only rats fed R-type of diets. Directly after the intervention period, both iron and iron and zinc supplementation caused a significant decrease in SOD and CAT activity in the liver, while after the post-supplementation period SOD activity was significantly lower and CAT activity was significantly higher in the iron and zinc supplemented rats than in the rats fed non-supplemented diet and diet supplemented with iron alone. The effect of combined iron and zinc supplementation on oxidative damage was also studied among young women [11], where the blood antioxidant status was impaired after iron supplementation and then was improved after combined iron and zinc supplementation period. The protective effect of zinc against to oxidative damage caused by high doses of iron can be explained by its role in body’s antioxidant status [7, 18, 19]. Zinc, as indirect antioxidant, reduces formation of free radicals as inhibitor of NADPH oxidase and an integral metal of Cu,Zn-SOD, induces metallothionein—a protein with antioxidant properties, and increases the protein sulfhydryl groups stability [20, 21]. Not only is the adequate zinc status important to a proper antioxidants defence against oxidative damage but the proportion of zinc to other minerals including iron is significant. In the presented studies, the proportions of Fe:Zn amounted 1:1 in iron and zinc

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supplemented diets and 10:1 in diets supplemented with iron alone. However, in spite of the proportion, the effect of iron supplementation on zinc concentration in the liver was not observed. In contrast, the results of other studies highlighted that in situation when Fe:Zn ratio was below 2:1, the iron did not significantly impair zinc absorption, but in the situation of the higher ratios, the zinc absorption might be significantly reduced [22]. The results of several studies on influence of combined iron and zinc supplementation on iron status are inconsistent [10, 11, 23–31]. In our studies, combined iron and zinc supplementation influenced iron status in the rats fed D and R diets similarly to iron supplementation alone. Also other authors did not observe significant impact of zinc addition to the diets on iron status parameters in rats [23–25] and humans [11, 26, 27], while Sreedhal et al. [10] found that the combined iron and zinc supplementation improved iron status, but less effectively than iron supplementation alone. In contrast, the results of some animal [28] and human [29– 31] studies demonstrated that the addition of zinc decreased efficiency of iron supplementation. The differences in the study findings may be the effect of different nutritional status, different doses of minerals used in experiments as well as another length of intervention period. While 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. In the rats fed D and R diets, the prolonged effect of iron and iron and zinc supplementation on iron concentration in the liver was observed. We can conclude that iron or iron and zinc supplementation used in our studies effectively complemented the iron content in the liver in D and R rats and hepatic iron overload was not induced. Lack of the similar prolonged effect in the rats fed C-type of diets is not surprising and seems logical with regard to the control of iron metabolism. Moreover, the prolonged effect of diet intervention on antioxidant status concerned especially the rats fed R-type of diets and was observed for such parameters as SOD and CAT activity, 8-isoprostane, protein carbonyl groups and 8-OHdG levels. The post-supplementation observation was particularly important for the evaluation of the protein oxidative damage due to the fact that the effect of used supplementation was delayed and the differences between groups of rats fed various types of diet reduced in all vitamins and minerals were visible only after this stage. The practical aspect of our research was to include groups of rats fed a diet with reduced amounts of all vitamins and minerals to the experiment. Although the reduction in all vitamins and minerals in the diet did not affect the measures such as body mass, haematology parameters and lipids profile (data not presented), the effect

Eur J Nutr

of applied dietary intervention on liver antioxidative status in those rats was higher than in the rats fed the adequate diet and/or in case of isolated iron deficiency. It shows that in a situation even of moderate deficiency of many nutrients it should be considered to supplement not only one but all of them. Moreover, it is important to asses the nutrients intake and nutritional status before introducing dietary supplements and monitoring body’s status during this treatment. Not only introducing a group of rats fed diets moderately deficient in vitamins and minerals and testing the effect of supplementation after the termination of this treatment but also comprehensive determination of the liver antioxidant status was the strength of our studies. We used numerous parameters, which included specific measurements of oxidative status such as 8-isoprostane or 8-OHdG. Conducting the experiments in controlled conditions allowed to eliminate confounding factors that might affect the results. On the other hand, feeding animals with semi-synthetic diets and hosted in controlled conditions are limitations in transferring the results to humans. Moreover, due to the fact that the experiment was conducted in two separate studies, it was not possible to perform the statistical analysis between group of rats fed C-type diets and a corresponding group of rats fed R-type diets.

Conclusions In summary, the findings of these animal studies indicate that zinc supplementation simultaneous with iron supplementation can protect liver against oxidative damage induced by high doses of iron in rats fed iron-deficient diet and diet with a reduced amount of all vitamins and minerals. The protective effect of this supplementation was observed even two weeks after the termination of this intervention. The post-intervention observation was relevant due to the fact that for examined parameters the effect of used intervention may be delayed and visible only after this period. Acknowledgments The studies were 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. Author contribution JK: study concept and design, oversaw the animal and diet manipulations; JK and DM: conducted the animal experiments; JK, DM and BP: assisted with tissue collection; JK, AR and ES: perform analytical determinations of study parameters; JK: performed the statistical analysis and drafted the manuscript; JK: analysis and interpretation of data; JK and BP: critical revision of the manuscript.

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The effect of iron and zinc supplementation and its discontinuation on liver antioxidant status in rats fed deficient diets.

The aim was to investigate the effect of iron or combined iron/zinc supplementation on rat liver antioxidant status...
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