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iSSn 2413-6077. iJmmR 2017 Vol. 3 issue 1 V. V. Ilika

dOI 10.11603/IJMMR.2413-6077.2017.1.7051

mORPhOLOGIcAL REsEARch ON fREE-REsIdUE OXIdATION 
PROcEssEs IN cAsEs Of dEcIdUAL cELLs Of PLAcENTA  
IN chORIOAmNIONOTIs ANd BAsAL dEcIdUITIs cOmBINEd  
WITh IRON-dEfIcIENcY ANEmIA IN ThE PREGNANT

V. V. Ilika 
BUKOVINIAN STATE MEDICAL UNIVERSITY, CHERNIVTSI, UKRAINE

Background. The oxidative modification of proteins is lately pivotal to pathologists and it is a new way of 
research on different pathological conditions, as well as the diagnostics of inflammation processes in placenta.

Objective. The study was aimed at the research of nitro peroxides and establishing the specific features of 
oxidative modification of proteins in inflammation of placenta with iron deficient anaemia in the pregnant.

Methods. Сhemiluminescent and histochemical technique (with bromphenol blue on ‘acidic’ and ‘basic’ 
proteins according to Mikel Calvo) was applied.

Results. The intensity of nitro peroxides glow in chorioamnionitis and basal deciduitis increased in comparison 
with the samples of physiological and iron deficient anaemia gestation. At the same time in chorioamnionitis the 
glow intensity is higher than in basal deciduitis.

Due to the results of immune histochemical technique held while analysing the samples, together with 
chorioamnionitis and basal deciduitis the R/B increases and in basal deciduitis the rate, is probably, higher, than 
in chorioamnionitis. At the same time, the extent of oxidative modification of proteins in cases of inflammation 
with iron deficient anaemia in the pregnant is on the average higher than with no iron deficient anaemia in the 
pregnant.

Conclusions. High level of nitro peroxides in placentae basal plate in secundines inflammation, the increase 
in r/b rate, in other words the prevalence of ‘acidic’ proteins over ‘basic’ ones, is evidenced due to the increase 
of the intensity of oxidative modification processes of proteins in cases of deciduitis.

KEy wORDS: inflammation; placenta; anaemia; iron-deficiency.

Introduction
The formation of active forms of oxygen 

and oxidative modification of macromolecules 
is usual and important biological processes. But 
the creation of oxygenous residues excess 
could damage cells and facilitate the develop-
ment of many illnesses [1], and in certain cases, 
a molecular pathology of the organism [2].

the oxidative modification of proteins is 
lately pivotal to pathologists [3], and it is a new 
way of research on different pathological con-
ditions [4], as well as the diagnostics of inflam-
mation processes in placenta [5]. In this area 
the scholars used a range of techniques aimed 
at detection of nitro peroxides [6], as the most 

responsive and long lasting oxidants among 
free residues, and at estimation of the degree 
of protein oxidative modification in various 
structures of placenta [7]. However, this aspect 
in combination with iron deficient anaemia in 
the pregnant has not been explored yet [8] and 
has been being studied that is the objective of 
our study.

Objective
Chemiluminescent technique with luminol 

was applied in the research of nitro peroxides 
in cases of inflammation. the aim of the study 
was to establish the specific features of 
oxidative modification of proteins in cases of 
deceduitis of basal plate in long lasting basal 
deciduitis and chorioamnionitis in combination 
with iron deficient anaemia and without it in 
the pregnant by means of immune histochemical 
and micro spectral photometric procedure.

Corresponding author:  Vitaliy Ilika, Department of Pathologic 
Anatomy, Bukovinian State Medical University, 15 Chelyuskintsev 
Street, Chernivtsi, 58000.
Phone number: +380951477073
E-mail: ilika.vitaliy@mail.ru

International Journal of Medicine and Medical Research 
2017, Volume 3, Issue 1, p. 75–78
copyright © 2017, TSMU, All Rights Reserved



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iSSn 2413-6077. iJmmR 2017 Vol. 3 issue 1

Methods
The control group consisted of placenta 

(n=20) received at the urgent delivery of clini-
cally healthy women in childbirth and in cases 
of iron-deficiency anaemia in gravidas (n=21). 
Experimental group consisted of the placenta 
(n=123) received from women in childbirth at 
gestation period of 37-40 weeks with chorioam-
nionitis and basal deciduitis combined with iron 
deficiency anemia in gravidas and without it.

the first procedure (a chemiluminescent 
technique) was performed on the frozen sec-
tions of placenta. The luminol originated che-
miluminescence was studied under the lumi-
nescent microscope LUMAM P8. On digital 
photomicrographies, obtained by means of 
computer estimation of glow intensity in a 256 
gradations scale: from 0 (glow absence indi-
cator) to 255 (glow top intensity), the quantitative 
measuring of luminescence was carried out [8].

For validity estimation of averages contrast 
Student’s t-test was used, after positive verifi-
cation of the samples for normal distribution 
in it was determined by the Shapiro-Wilk test 
(computer program PAST 3. 14, free license, 
O.  Hammer, 2016) [9]. For approval or rejection 
of statistic hypothesis the significant difference 
p≤0.05 was used.

to evaluate the extent of oxidative modi fi-
cation of proteins, Histochemical technique was 
carried out with bromphenol blue on ‘acidic’ 
and ‘basic’ proteins according to Mikel Calvo 
[10].

Optic images were converted into digital 
using the digital camera and were analysed 
with licensed copy of the ImageJ computer 
program (1.48, W. Rasband, National Institute 
of Health, USA) using probe computer spec-
trometry in the system of colour analysis RGB. 
According to the designated system of colour 
analysis the intensity of red and blue colours in 

coloration was assessed. According to the Mikel 
Calvo technique, red coloration matches car-
bonyl groups, whereas blue coloration matches 
protein amines; therefore establishment of 
mathematical relation between coloration 
intensity in two colours (spectrum areas) the 
R/B was received and the extent of oxidative 
modification of proteins was evaluated (relation 
of carboxylic and amines) [11,12].

Results
Chemiluminescent glow of nitro peroxides 

was evaluated and the level of physiological 
gestation of placentae was estimated in cases 
of iron deficient anaemia in the pregnant. the 
averages are introduced in Table 1.

The results of quantitative indicators of 
chemiluminescent glow of nitro peroxides and 
the R/B rate in cases of deciduitis of placenta 
basal plate in acute and long lasting chorio-
amnionitis, and basal deciduitis in combination 
with iron deficient anemia in the pregnant are 
presented in Table 2.

Discussion
According to the data above, we should 

emphasise that in physiological gestation of 
pregnancy in cases of iron deficient anaemia, 
significant deviations in any conducted 
technique was not evidenced. The data in Table 
2 proves that in chemiluminescent research 
technique the intensity of nitro peroxides glow 
in cases of chorioamnionitis and basal deciduitis 
increases in comparison with the samples of 
physiological and iron deficient anaemia 
gestation. The fact that, in cases of placentitis 
with underlying iron deficient anaemia in the 
pregnant, the quantitative indicators are higher 
than with no anaemia. At the same time, in 
cases of chorioamnionitis, the glow intensity is 
higher than in basal deciduitis. 

Table 1. The quantitative indicators of chemiluminescent nitro peroxide glow and R/B rate 
(histochemical technique on ‘acidic’ and ‘basic’ proteins with bromphenol blue according  

to Mikel Calvo) in cases of deciduitis of placenta basal plate in physiological gestation  
and iron deficient anaemia in the pregnant

Basal pate 
structures

Research groups

Observation of physiological gestation
(n=20)

Observation of iron deficient anemia  
in the pregnant without inflammation  

of secundines
(n=21)

Decidual 
cells of ba-

sal plate

Chemilumi nes-
cent glow of 

nitro peroxides 
with luminol

Histochemical technique 
on ‘acidic’ and ‘basic’ 

proteins with bromphenol 
blue on Mikel Calvo

Chemilumi nes-
cent glow of 

nitro peroxides 
with luminol

Histochemical technique 
on ‘acidic’ and ‘basic’ 

proteins with bromphenol 
blue on Mikel Calvo

34±3.8 1.04±0.008 38±4.2
p>0.05

1.06±0.009
p>0.05

V. V. Ilika



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The photomicrographs of histological ima-
ges give an idea of how some structures of 
placental basal plate look like, decidual cells in 
particular, when the immunohistochemically 
technique for ‘acidіc’ and ‘basic’ proteins 
according to Mikel Calvo is applied (Fig.1).

When assessing visual histochemical 
preparations, decidua cells are clearly stained 
that is applicable for quantitative research, cell 
boundaries are defined by clear cell membrane 
colouring and contrasting colour around 
decidua cells fibrinoid. nuclei and nucleoli were 
visualized fairly well. ‘Basic’ proteins prevailed 
in nucleoplasm, while ‘sour’ in the nucleolus.

the decidua cells cytoplasm specific colour 
was mostly granular in nature and spectral 
characteristics and optical density of colour 
varied greatly.

Relatively invariable data in assessing the 
degree of oxidative modification of proteins in 

placentas of physiological gravidity and gra-
vidas are observed, which can be interpreted 
as the manifestation of involutory changes in 
placenta. The R/B factor is only slightly higher 
than what can be evaluated as a slight predo-
minance of ‘sour’ proteins over ‘basic’ ones. 
these figures are important in terms of the R/B 
factor assessing, as an indicator of oxidative 
modification of proteins. however while ana-
lysing the samples with chorioamnionitis and 
basal deciduitis the R/B increases and in basal 
deciduitis the rate is probably higher than in 
chorioamnionitis. At the same time, the extent 
of oxidational modification of proteins in cases 
of inflammation in combination with iron de-
ficient anaemia in the pregnant is on the ave-
rage higher than with no iron deficient anaemia 
in these patients.

taking into account our findings of high 
level of nitro peroxide in basal lamina of placen-

Table 2. Quantitative indicators of chemiluminescent glow of nitro peroxides and the R/B rate 
(histochemical technique on ‘acidic’ and ‘basic’ proteins with bromphenol blue on Mikel Calvo)  

in cases of deciduitis of placenta basal plate in acute and long lasting chorioamnionitis  
and basal deciduitis in combination with iron deficient anemia in the pregnant

Research 
groups

Chemiluminescent glow of nitro peroxides 
with luminol

Histochemical technique on ‘acidic’ and 
‘basic’ proteins with bromphenol blue on 

Mikel Calvo R/B rate
(M=m)

Secundines 
inflammation

Secundines inflammation 
in cases of iron deficient 
anaemia in the pregnant

Secundines 
inflammation

Secundines inflammation 
in cases of iron deficient 
anaemia in the pregnant

Acute 
chorio-

amnionitis

154±4.9
(n=20)

186±5.1
(n=21)

p=0.003

1.24±0.011
(n=20)

1.64±0.016
(n=21)

p<0.001
Acute basal 
deciduitis

130±4.4
(n=21)

164±4.5
(n=20)

p=0.002

1.89±0.015
(n=21)

2.14±0.018
(n=20)

p<0.001

fig. 1. Photomicrograph of placental basal plate. Нistochemical technique for ‘acidic’ and ‘basic’ proteins with bromphenol 
blue according to Mikel Calvo. On the right – observation during physiological pregnancy. On the left – in cases of chronic 
deciduitis in placental basal plate.

 

  
| 50 micrometers | | 50 micrometers | 



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tas which are characterized by secundines in-
flammation, increase in, i.e. the prevalence of 
‘sour’ proteins over ‘basic’ ones; correlation 
between the increasing intensity of oxidative 
modification of proteins in cytoplasm of deci-
duitis and secundines inflammation would be 
reasonable.

Conclusions
Due to the chemiluminescent technique of 

nitro peroxides evaluation and immune histo-
chemical technique of ‘acidic’ and ‘basic’ pro-
teins assessment in cases of deciduitis of 

placenta basal plate, the impetuous increase of 
indicators is observed.

the inflammation of secundines in com-
bination with iron deficient anaemia in the 
pregnant was evidenced by higher average 
rates than with no anaemia. 

Considering the received data that proves 
high level of nitro peroxides in placentae basal 
plate in secundines inflammation, the increase 
in R/B rate, in other words prevalence of ‘acidic’ 
proteins over ‘basic’ ones, is evidenced due to 
the increase of intensity of oxidative modification 
processes of proteins in cases of deciduitis.

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Received: 2017-01-17

V. V. Ilika