EJBR2018v8i3art168-173


ISSN 2449-8955 
European Journal  

of Biological Research 
Research Article 

 

European Journal of Biological Research 2018; 8 (3): 168-173 

 

In vitro studies of iron absorption and activity of 

glutathione peroxidase in intestinal mucosa of the chicken 

 

J. Markovs
1
, A. Galuza

1
*, N. Basova

2
, G. Knipse

1
, S. Vasiljeva

2
, G. Smirnova

2
 

  
1
 Department of Anatomy and Histology, Faculty of Medicine, University of Latvia, Riga, Latvia 

2
 Institute of Biology of the University of Latvia, Riga, Latvia 

*Corresponding author: Agate Galuza; E-mail: agate.galuza@gmail.com 

 

 

 

ABSTRACT 

 

We examined the absorption of iron, the activity of 

selenoprotein glutathione peroxidase (GSH· Px) and 

cellular compartmentalization of metal in the 

chicken duodenum and ileum. The method of 

accumulating mucosa preparation (AMP) was used. 

It was shown that the intestinal iron accumulation is 

dose-dependent process, which has two components: 

transcellular and paracellular. The realization of 

these pathways is region-specific and depends on 

exposed iron levels. Slightly elevated iron status of 

intestinal mucosa does not influence activity of 

GSH· Px. At the same time the results indicate that 

the activity of glutathione peroxidase can be altered 

by iron overload. Immunohistochemistry revealed 

that stainable iron could be co-localized to the 

endolysosomal compartment. How the activity of 

enzyme can be affected by oxidative stress and 

competitive interactions of iron with selenium are 

discussed. 

 

Keywords: Glutathione peroxidase; Iron absorption; 

Intestinal mucosa; Chicken. 

 

1. INTRODUCTION 

 

 Iron serves numerous functions in the body 

relating to the metabolism of oxygen. Ferrous iron 

can react with oxygen to form superoxide and             

also can homolytically cleave hydrogen peroxide 

yielding hydroxyl radicals and hydroxyl ions. These 

ions are particularly aggressive and elicit toxic 

effects, which are mainly related to oxidative stress 

[1]. Moreover, iron is deeply linked to cell death 

pathways through reactive oxygen species (ROS) 

production [2]. Therefore most of free iron is safely 

stored in a non-redox-active form in ferritins. Iron 

overload is strongly associated with the intensi-

fication of free radical oxidation [3]. Glutathione is a 

main detoxifier of ROS in the intestine. Glutathione 

peroxidase provides detoxification of peroxides by 

using reduced glutathione, and is one of the most 

important antioxidant enzymes [4]. GSH· Px is a 

selenoprotein, and selenium availability regulates 

glutathione peroxidase enzyme activity [5]. An 

excess of certain minerals in the body can anta-

gonize other minerals and cause depletion [6].  

 Since animals lack mechanisms for iron 

elimination, iron uptake is strictly regulated. The 

non-heme iron is ultimately taken up from the gut 

lumen by divalent metal transporter 1 (DMT1) situ-

ated on the microvillus membrane, before joining 

the labile iron pool in the cytoplasm and transferred 

to the bloodstream by ferroportin 1 [7]. The mecha-

nism and regulation of intestinal iron absorption are
 

incompletely understood in spite of their pivotal role 

in the maintenance of body iron homeostasis [8]. 

Received: 27 May 2018; Revised submission: 22 July 2018; Accepted: 08 September 2018 
Copyright: © The Author(s) 2018. European Journal of Biological Research © T.M.Karpiński 2018. This is an open access article  

licensed under the terms of the Creative Commons Attribution Non-Commercial 4.0 International License, which permits  

unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. 

DOI: http://dx.doi.org/10.5281/zenodo.1412784 



169 | Markovs et al.   Iron absorption and activity of glutathione peroxidase in intestinal mucosa of the chicken 

European Journal of Biological Research 2018; 8 (3): 168-173 

 

Iron metabolism involves iron trafficking along 

specific cellular compartments, including endoso-

mes and lysosomes [9]. These organelles take center 

stage in cellular iron accumulation and are involved 

as a control hub for aging and longevity [10]. The 

study described here was undertaken to investigate 

the influence of exposure to iron in concentrations 

occurring in contaminated food and feed on 

accumulation and compartmentalization of iron in 

enterocytes and the activity of GSH· Px in the 

intestinal mucosa. 

 

2. MATERIALS AND METHODS 

 

2.1. Animals and experimental design 

 

 New-hatched Lohmann brown cockerels were 

obtained from the Latvian poultry company 

BALTICOVO. All of the experimental procedures 

were approved by the Animal Ethics Committee of 

the Food and Veterinary Service (Riga, Latvia, 

authorisation reference number 13, from December 

22, 2008). The chickens were housed in cage units 

with free access to food and water. Animals received 

standard full-feed diet. For the in vitro study 30  

days old chickens were divided into 3 groups (5 in 

each group): 1 - “Buffer”, 2 - “+ Fe 0.512 mM                 

as iron sulfate”, 3 - “+ Fe 2.56 mM as iron sulfate". 

Chickens were sacrificed by decapitation, in accor-

dance with recommendations for the euthanasia      

of experimental animals of the European Conven-

tion [11]. 

 

2.2. Determination of iron absorption and GSH-

Px activity 

 

 The content of iron in chick intestinal mucosa 

was estimated by atomic absorption spectrophoto-

metry [12], the activity of glutathione peroxydase 

(GSH· Px) - by a modified Pinto-Bartley method [13]. 

 The intestine was isolated and washed with 10 

ml of cooled physiological solution (154 mM NaCl). 

Then it was placed on ice-cold glass plate. Duo-

denum and ileum were cut on segments (5 cm) and 

used for intestinal preparations. Iron binding by the 

intestinal wall was studied by means of AMP 

method as developed by Ugolev et al. [14] for 

investigation of the first stages of transport 

processes. An everted intestinal segment of birds 

belonging to groups 1, 2 and 3, mounted on a glass 

rod, was submerged in 7 ml Tris-buffer containing 

different concentration of iron (0.512 mM and             

2.56 mM). An everted intestinal segment of birds 

belonging to groups 1, 2 and 3, mounted on a glass 

rod, was submerged in 7 ml Tris-buffer containing 

different concentration of iron (0.512 mM and 2.56 

mM). 

 Intestinal AMP were incubated for 30 min at 

41ºC. Tris-buffer without iron supplement was         

used as a control. Buffer composition (mM) was:            

4 Tris hydrochloride, 145 sodium chloride, 4 potas-

sium chloride, 20 fructose, pH 7.4. The amount of 

accumulated iron was calculated as the difference 

between the iron contents in the mucosa before and 

after incubation. 

 

2.3. Histological examination 

 

 For histological examination, 1-cm segments 

of intestinal samples from animals of the 2-nd and  

3-rd group (duodenum was taken 0.5 cm distal to  

the ampulla of Vater and ileum - 10 cm proximal to 

the ileocecal junction) were isolated and fixed in 

10% neutral buffered formalin. Paraffin-embedded 

tissue was cut into 4-µ m-thick sections and stained 

with haematoxylin-eosin and the periodic acid-

Schiff (PAS) reagent. Duodenal sections were 

colored with Perls’ Prussian blue stain for iron 

detection. Late endosomes and lysosomes in the 

enterocytes were highlighted by immunohisto-

chemistry using an anti-CD68 and anti-TRPV1 

antibodies. 

 

2.4. Statistical analysis 

 

 All statistics were performed using the 

program SPSS. Means and standard deviations and 

significance values were calculated. The results 

were assessed statistically by t tests. Statistical signi-

ficance was set at p<0.05. 

 

3. RESULTS AND DISCUSSION 

 

 Iron exercised a diversified action: after 30 

min incubation in a medium containing 0.512 mM 

of iron its concentration in the duodenal mucosa 

amounted to 9.72 ppm, and the iron accumulation 

was increased by 57.3% (Table 1). At the same time 



170 | Markovs et al.   Iron absorption and activity of glutathione peroxidase in intestinal mucosa of the chicken 

European Journal of Biological Research 2018; 8 (3): 168-173 

 

in the ileal mucosa exposed to lower level of iron 

only nearly 10% of the metal was accumulated.          

The obtained data indicated 5.8-fold ability of the 

duodenum, compared with the ileum, to transfer iron 

into the mucosa. After applying of 5-times higher 

iron concentration in the incubation medium the 

tissue level of this metal increased more than two 

times and the metal accumulation in the duodenal 

mucosa was increased by 133.8% vs. 330.7% in          

the ileal mucosa. Dramatic effects of higher iron 

exposure on accumulation of this metal in the ileal 

mucosa with levels more than 30-fold higher than 

observed for lower levels of iron exposure may be 

related to greater (paracellular) leakiness of the 

epithelial barrier in the ileum. It is known, that 

transcellular active transport of iron across the gut 

epithelium occurs mainly in the duodenum and 

jejunum [15]. We conclude that similar to calcium 

absorption, passive, paracellular absorption of iron 

predominates in the ileum when dietary iron levels 

are high [16]. 

 

 

Table 1. Iron accumulation in intestinal mucosa of chickens. 

Experimental 

conditions 

Concentration of Fe in intestinal mucosa, 

ppm 

Accumulation of Fe in intestinal mucosa, 

ppm 

Duodenum Ileum Duodenum Ileum 

1. Buffer 6.18 ± 0.88 3.02 ± 0.30 - - 

2. +Fe (0.512 mM) 9.72 ± 0.65
a
 3.36 ± 0.29 3.54  (+57.3% ) 0.32 (+9.9%) 

3. + Fe (2.56 mM) 14.40 ± 1.50
a,b 

14.00 ± 1.52
a,b

 8.22  (+133.8%) 10.98  (+330.7%) 
a
Statistically different from the 1

st
 group (P<0,05); 

b
Statistically different from the 2

nd
 group (P<0,05) 

 

 

 As revealed by our studies, the activity of 

GSH· Px in the 2nd group either remains unaffected 

(in the duodenal mucosa), or decreases insigni-

ficantly (in the ileal mucosa), but in the 3rd group 

both in the duodenum and ileum a statistically 

significant decreasing trend in GSH· Px activity was 

observed with increasing iron accumulation in 

intestinal mucosa (Table 2).  

 

 

Table 2. Activity of GSH· Px in intestinal mucosa of 

chickens 

Experimental 

conditions 

Activity of GSH· Px µmol 

GSH/min/g 

 Duodenum Ileum 

1. Buffer 2.37 ± 0.24 1.62 ± 0.25 

2. +Fe  (0.512 mM) 2.36 ± 0.80 1.46 ±  0.63 

3. + Fe  (2.56 mM) 1.44 ± 0.38 
a,b

. 0.93 ± 0.31 
a
 

a 
Statistically different from the 1

st
 group (P<0.05)

 

b
Statistically different from the 2

nd
 group (P<0.05) 

 

 

 Stainable iron was found in the small 

intestinal enterocytes of the chickens in the 3
rd

 

group. As shown in Fig. 1-A, iron deposits appeared 

as a narrow string of punctae in the subapical area 

all along the brush border. Little or no diffuse 

staining of the enterocyte cytosol was detected. 

TRPV1 immunoreactivity was localized in the 

subapical compartment of the villous enterocytes, 

having a punctuate appearance (Fig. 1-B). The 

pattern of CD68 immunoreactivity was quite similar 

to selective cytoplasmic expression of TRPV1 (Fig. 

1-C). It should be emphasized, that both CD68 and 

TRPV1-positive material and iron deposits within 

enterocytes were consistently localized to the same 

area in the vicinity of the brush border. It is well 

known that the endosomal-localized DMT1 is 

responsible for mobilizing iron out of endosomes 

[17]. It was shown that members of the transient 

receptor potential (TRP) superfamily could function 

as intracellular cation release channels whose locali-

zation is commonly assigned to late endosomes and 

lysosomes [18]. The obtained results also indicated 

that TRPV1 is localized to the late endosomes and 

lysosomes, where TRPV1 may function to transfer 

the endosomal free Fe
2+

 into the cytoplasm in the 

transferrin cycle in parallel to DMT1.  

 Our data showed that chickens of the 3
rd

 

group had lower GSH· Px activity in the intestinal 

mucosa than did animals in the 1
st 

and 2
nd

 group. It is 

likely that in the 3
rd

 group iron reaches damaging 

levels, exceeding the homeostatic capacity of the 



171 | Markovs et al.   Iron absorption and activity of glutathione peroxidase in intestinal mucosa of the chicken 

European Journal of Biological Research 2018; 8 (3): 168-173 

 

enterocytes. Decreased GSH· Px activity has been 

reported in tissues where oxidative stress occurs in 

several pathological animal models [19]. It is 

known, that an excess of iron in tissues can induce 

hydroxyl radical formation. This effect was likely 

promoted by the recycling of chelated, inactive Fe
3+

 

to the active Fe
2+

 state by the Fenton reaction in the 

mitochondria [20]. Fe
2+

 is extremely toxic because  

it can rapidly react with hydrogen peroxide and 

molecular oxygen to produce reactive oxygen 

species. Proteins are oxidatively damaged by the 

combined action of free radicals and the trace metal 

ions such as Fe
2+

 and Cu
2+

 [21]. In our experiments
 

oxidative damage to GSH· Px may also affect its 

activity. The seeming paradoxical dissociation 

between considerable iron accumulation in the ileal 

mucosa in the 3
rd

 group and only moderate down-

regulation of GSH· Px activity comparable to that in 

the duodenal mucosa can be explained by the prefe-

rential use of paracellular route of iron transport 

under these circumstances. 

 
 

 
Figure 1. A - Iron histochemistry with Perls’ staining of 

chicken intestinal mucosa from a third group. Iron 

deposits in the subapical compartment of villous 

enterocytes, x40. B - TRPV1-positive punctae in the 

subapical compartment of villous enterocytes (arrows) 

x40. C - CD68 expression in the enterocytes with the 

subapical pattern (arrows). Likewise, high levels of  

CD68 expression are associated with macrophages 

(arrowheads), x40.  

 
 

 According to the reports, supplementary iron 

reduces selenium bioavailability [22]. Therefore, 

reduction of the activity of selenoprotein GSH· Px 

during the iron overload may be related at least in 

part to the competitive iron interactions with 

selenium, thus reducing its bioavailability.  

 Understanding of the ways and control of 

transition metal uptake and translocation is very 

important particularly because some of them can be 

highly toxic when accumulate in the cells. Our 

animal model of iron overload has demonstrated the 

accumulation of selective iron subapical deposits 

colocalized with endolysosomal markers. Labile 

iron can readily generate ROS, and sequestration in 

the endolysosomal apical system may represent one 

of many protective mechanisms that exist within        

the absorptive epithelial cell. These data seems to 

support the theory that at least half of the iron 

transported across the villous enterocytes uses a 

vesicular pathway and that a significant portion of 

the vesicular pathway involves the endolysosomal 

system, which is located en route towards the 

basolateral membrane [23, 24]. 

 Low iron levels may have a link with 

cognitive health later on in life. For example, the 

patients with anemia had a higher risk of developing 

dementia compared with those who were not anemic 

[25]. Eating foods high in iron can help prevent 

dementia. On the other hand, our results showed, 

that the iron supplementation can cause side effects 

and, consequently, compromise the life expectancy 

mainly for elderly populations, because age-related 

iron overload is a known contributor to multiple 

degenerative diseases, including cancer, liver fibro-

sis and heart attack [26-31].  

 

4. CONCLUSION 

 

 In conclusion, after iron treatment (0.512 mM 

in the incubation medium) GSH-Px activity remains 

unchanged despite the accumulation of metal in the 

intestinal mucosa. However pathological accumu-

lation of the iron within the intestinal mucosa (2.56 

mM in the incubation medium) elicits toxic effects, 

reducing the activity of GSH-Px, which are mainly 

related to oxidative stress. The endolysosomal com-

partment plays an important role in cellular iron 

homeostasis in the iron-overloaded state. 

 

AUTHORS’ CONTRIBUTIONS 

 

JM: Study design and interpretation of the protocol 

and guidance; acquisition of the data; obtained 

funding; drafting of the manuscript; critical revision 

of the manuscript for important intellectual content; 



172 | Markovs et al.   Iron absorption and activity of glutathione peroxidase in intestinal mucosa of the chicken 

European Journal of Biological Research 2018; 8 (3): 168-173 

 

AG and NB: Study concept and design; analysis and 

interpretation of the data; drafting of the manuscript; 

critical revision of the manuscript for important 

intellectual content; statistical expertise; study 

supervision. GK, SV, GS: analysis and interpretation 

of the data; drafting of the manuscript; critical 

revision of the manuscript for important intellectual 

content; administrative, technical and material 

support. All authors read and approved the final 

manuscript. 

 

CONFLICTS OF INTEREST 

 

The authors declare that there is no conflict of 

interest regarding the publication of this article. 

 

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