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iSSn 2413-6077. iJmmR 2016 Vol. 2 issue 2

DOI 10.11603/Ijmmr.2413-6077.2016.2.7034

LIPID PeroxIDatIon In muLtIPLe organ faILure  causeD by 
assocIateD chest anD hIP trauma

M. I. Marushchak, M. M. Khudobіak, I. Ya. Krynytska, I. V. Antonyshyn
I. HorbacHevsky TernopIl sTaTe MedIcal UnIversITy, TernopIl, UkraIne

Background. The injured with polytrauma are classified as extremely severe patients, as their emergency 
care and intensive care is followed by significant difficulties, frequent development of complications, high mortality 
and survivors disability.

Objective. This study is aimed to evaluate the effect of lipid peroxidation in heart, lungs, liver and blood of 
rats in early and late periods of combined traumatic injury of chest and hips and to establish their influence on 
the development of multiple organ failure.

Methods. The study was conducted on 70 adult male white nonlinear rats. It was determined the content 
of lipid hydroperoxides and concentration of TBA-active products in erythrocyte mass, heart, lungs and liver.

Results. The data prove activation of free radical oxidation at the first day of post-traumatic period. Analysis 
of the data evidences the increase in of lipid hydroperoxides (HPL) rate in liver homogenate in group E1 in 1.5 
times, in all subsequent periods of the research the increase in rate fluctuated within 1.9-2.0 times. In blood and 
heart the HPL rate increased twice in group E1, reached maximum in group E2 and gradually decreased till the 
end of the experiment, data exceeded the control group. The highest level of HPL was determined in lung tissues 
(r≤0.01). In the post traumatic period the highest TBA-AP was detected in lungs and liver.

Conclusions. In case of simulated trauma (injury of chest with fractures of both hips) hyperactivation 
processes of free radical oxidation is observed in 1 day, reaching a peak in 7–14 days of post-traumatic period 
in blood, tissues, liver, heart and lungs if compared to the control group causing multiple organ failure.

KEY WORDS: trauma, lipid peroxidation, lung, liver, heart.

Introduction 
Nowadays one of the most urgent problems 

of medicine are multiple and combined injuries 
[1, 2]. This is due to the constant increase of the 
number of multisystem and multiorgan injuries 
on the background of some general manifesta­
tions of injuries stabilization, which leads to 
mortality increase [3, 4]. As a result of constant 
technological advances the increase in the 
number of man­made disasters has led to a 
significant increase in the number of patients 
with polytrauma. According to WHO trauma is 
one of the top five causes of death among 
people aged 15–44; mortality from injuries and 
accidents takes the first place [5]. The injurees 
with polytrauma are classified as extremely 
se vere patients, as emergency care and inten­
sive care is accompanied by significant diffi­

culties, frequent development of complications, 
high mortality and survivors’ disability [6]. In 
case of multiple trauma, wounded are primarily 
affected by phenomena of traumatic­haemor­
rhagic shock and tissue hypoxia. In the post­
traumatic period unnoticed injuries, infection, 
SIRS, sepsis and multiple organ failure threaten 
their life and health [7].

Therefore, our study was aimed to evaluate 
the effect of lipid peroxidation in heart, lungs, 
liver and blood of rats in early and late periods 
of combined traumatic injury of chest and hips 
and establish their influence on the development 
of multiple organ failure.

Material and Methods 
The study was conducted on 70 adult male 

white nonlinear rats of 200–220 g body weight. 
Rats were housed under standardized labo­
ratory conditions, with 12 h dark/light cycle and 
free access to food and tab water ad libi tum. 
All procedures were conducted according to 
the European Convention for the Protection of 

Corresponding author: Marya Marushchak, Department of 
Functional Diagnostics and Clinical Pathophysiology, I. Hor-
bachevsky Ternopil State Medical University, 1 Maidan Voli, 
Ternopil, Ukraine, 46001 
Phone number: +380979901202 
E-mail: marushchak@tdmu.edu.ua 

International Journal of Medicine and Medical Research 
2016, Volume 2, Issue 2, p. 52–55
copyright © 2016, TSMU, All Rights Reserved

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Vertebrate Animals used for Experimental and 
Other Scientific Purposes (Strasbourg, 1986) 
and General Ethical Principles of Experiments 
using Animals (First National Congress of Bio­
ethics, Kyiv, 2001). The animals were randomly 
divided into 8 groups: 1 control (C1) and 5 expe­
rimental (E1, E2, E3, E4, E5) groups, each 
comprising 12 animals. Rats of experimental 
groups. The animals in the experimental group 
underwent thiopental sodium anaesthesia 
(40 mg/kg intraperitoneal rat) using a trocar 
modeled right­closed pneumothorax from frac­
tured ribs and combined them with a broken 
left and right femur. Skeletal injury was mo­
deled by applying a single shock dosed by a 
specially designed device on each thigh, which 
caused a closed fracture [8]. Impact energy was 
0.375 J, which corresponded to the injury of 
mo derate severity. Combined injury was mode­
led by sequential administration of these two 
injuries. Mortality of animals in each group was 
E1(12/11), E2(12/9), E3(12/10), E4(12/10), 
E5(12/9). At the end of the experimental period, 
the rats were sacrificed by decapitation.

The content of lipid hydroperoxides in 
erythrocyte mass, heart, lungs and liver was 
determined by [9]. This method is based on 
spectrophotometrical optical density measu­
rement of the products of ammonium thiocya­
nate, hydrochloric acid and Mohr salt reaction. 
Lipids from the samples were preliminarily 
extracted with ethanol. Selection of tissue 
samples and preparation for extraction were 
performed at 4°C. Ethanol (2.8 ml) and 0.05 ml 
of 50% trichloroacetic acid (TCA) were added to 
0.2 ml of hemolisate (dissolved in buffer solu­
tion with pH 7.4), and shaken for 5–6 min. 
Obtained protein precipitate was separated by 
centrifugation at 700 g. Ethanol (1.2 ml), 0.02 ml 

of concentrated HCl, and 0.03 ml of 1% Mohr 
salt solution in 3% HCl were added to 1.5 ml of 
supernatant. The mixture was stirred. After 
30 s, 0.2 ml of 20% ammonium thiocyanate was 
added, and then the absorbance of the solution 
was determined at λ=480 nm. In a control 
sample, the appropriate amount of bidistilled 
water was added instead of supernatant. The 
content of lipid hydroperoxides was calculated 
by the difference between experimental and 
control values, and expressed in arbitrary units 
of optical density for 1 mg of protein.

The concentration of TBA­active products, 
characterizing the LPO rate, was assessed by 
Korobeinikova method based on the reaction 
between malondialdehyde (MDA) together with 
other products of peroxidation and thiobar­
bituric acid (TBA), occurring at high temperature 
and in acidic environment, and forming the 
colored complex of one MDA and two TBA mo­
lecules [10]. Protein concentration was measu­
red by Lowry [11]. All reagents used were 
obtained from SigmaAldrich and Fluka (USA). 
The experimental data were processed by 
variation statistics methods by the OriginPro 8 
program. Student t­test was used to determine 
the likely differences between the means of the 
samples. In all cases, reliable differences were 
considered by P value under 5% (P<0.05).

Results and Discussion
The data prove activation of free radical 

oxidation at the first day of post­traumatic 
period (Table 1 and 2). Activation of peroxide 
oxidation is one of the factors destabilizing 
membrane [12]. It is established that the action 
of hydroxyl radicals in diene conjugates fatty 
acids, lipid hydroperoxide is formed, which 
cause conformational irregularities in cell mem­

Table 1. The rates of lipid hydroperoxides in rat tissues (cu/mg protein)  
after combined trauma of chest and both hips (M±m)

Animal group Blood Heart Lungs Liver
Control 0.98±0.05 0.83±0.02 0.63±0.02 0.83±0.02
Trauma of chest and hips, 
1st day of observation

1.96±0.01*
р≤0.01

1.64±0.02*
р≤0.01

1.62±0.01*
р≤0.01

1.26±0.01*
р≤0.05

Trauma of chest and hips,
3rd day of observation

2.36±0.08*
р≤0.05

2.20±0.07*
р≤0.01

1.79±0.01*
p≥0.05

1.58±0.02*
р≤0.01

Trauma of chest and hips,
7th day of observation

2.03±0.06*
р≤0.01

1.82±0.01*
–

2.04±0.05*
р≤0.01

1.80±0.01*
р≤0.01

Trauma of chest and hips, 
14th day of observation

1.88±0.06*
–

1.78±0.02*
–

1.85±0.01*
р≤0,01

1,65±0.01*
р≤0,05

Trauma of chest and hips, 
28th day of observation

1.71±0.04*
–

1.62±0.04*
–

1.62±0.01*
р≤0.01

1.57±0.02*
р≤0,01

Notes. Tables 1 and 2: * — significant differences between the baseline indicators of the studied and control groups; 
p — significant differences between the experimental groups.

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branes. Analysis of the data evidences the 
increase in HPL rate in liver homogenate in 
group E1 in 1.5 times, in all subsequent periods 
of observation the increased rate fluctuated 
within 1.9–2.0 times. In blood and heart the HPL 
rate increased twice in group E1, reached a 
maximum in group E2, and gradually decreased 
until the end of the experiment, data exceeded 
the control group (Table 1). The highest level 
of HPL was determined in lung tissues, parti­
cularly in E1 it exceeded in 2.6 times the control 
rate, respectively E2 — in 2.9 times, E3 — in 3.2 
times, E4 — in 3.0 times and E5 — in 2.5 times 
(r≤0.01). Due to the fact that in case of trauma 
in lung tissues the activation of neutrophils is 
the main source of reactive oxygen species [13], 
and taking into account the administered chest 
injury, we believe that the pathological pro­
cesses that occur in lungs in case of simulated 
injuries have a significant contribution to the 
development of multiple organ failure.

It is established that fatty acids in the 
ground connection break into fragments of 

aldehyde groups at the ends to form malonic 
dialdehyde. Analysing the change of active 
products thiobarbituric acid in rat tissues, dif­
ferent activity of lipid peroxidation in the stud­
ied rat organs was revealed (Table 2). The post 
traumatic period following combined chest 
trauma of both thighs on the 1st day surveillance 
the highest TBA­AP was detected in lungs and 
liver, respectively, in 3 days — in blood and 
lungs, in 7 days and in subsequent periods of 
observation — in blood and liver (Table 2).

For a comparative analysis of the flow of 
free radical oxidation in various organs combi­
ned chest trauma of both thighs was adminis­
tered, the control group was regarded as 100%. 
It is established that the phenomenon of lipid 
peroxidation in heart was the most intense on 
the 3rd day after polytrauma simulation, respec­
tively, in lungs — from the 3rd to the 7th day, in 
blood — on the 7th day, in liver — on the 14th 
day (Fig. 1). Scientists have proved that the 
maxi mum intensity of lipid peroxidation coin­
cides with the body's response to inflammation 

Table 2. The rate of active products of thiobarbituric acid in rat tissues (mmol/mg protein)  
after combined trauma of chest and both hips (M±m)

Animal group Blood Heart Lungs Liver
Control 0.22±0.01 0.36±0.01 0.20±0.08 0.40±0.02
Trauma of chest and hips,  
1st day of observation

0.40±0.01
p≥0.05

0.70±0.02*
р≤0.01

0.42±0.02
p≥0.05

0.87±0.02*
р≤0.01

Trauma of chest and hips,  
3rd day of observation

0.75±0.02*
р≤0.05

0.97±0.03*
р≤0.01

0.68±0.02*
р≤0.01

1.23±0.02*
р≤0.01

Trauma of chest and hips,  
7th day of observation

1.06±0.03*
р≤0.01

0.80±0.02*
p≥0.05

0.67±0.01*
p≥0.05

1.76±0.03*
р≤0.01

Trauma of chest and hips,  
14th day of observation

0.86±0.02*
р≤0.01

0.75±0.02*
p≥0.05

0.60±0.02*
р≤0.01

2.08±0.04*
р≤0.05

Trauma of chest and hips, 
28th day of observation

0.72±0.03*
p≥0.05

0.68±0.03*
p≥0.05

0.53±0.02*
p≥0.05

1.87±0.02*
p≥0.05

Fig. 1. Severity of the processes of lipid peroxidation (%) in rat tissues (as example, MDA­AP) in post­traumatic period 
following combined trauma of chest and both hips.

0

100

200

300

400

500

600

1 day 3 day 7 day 14 day 28 day

blood
heart

lung
liver

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[14]. The development of hypoxemia caused by 
chest trauma affected lipid peroxidation sti­
mulation causing disruption of cell membranes 
structure and consequently cell death closing 
the ‘vicious’ circle, which in our and other 
authors opinion cause multiple organ failure 
with involvement of heart, lungs, liver in the 
pathological process [15].

The data indicate that the intensity of lipid 
peroxidation in different tissues of body de­
pends on injury type. Thus, Kozak D.V. in the 
research [16] notes that the intensity of lipid 
peroxidation due to blood loss and hip trauma 
has oscillatory nature of the duration increase 
up to the third day, the period of temporary 
welfare in 14 days and repeated increase in 21 

days. Our results prove that various tissues 
experience peroxidation activation and its 
maximum occurs at different time.

Conclusions
In case of simulated trauma (chest injury 

with fractures of both hips) hyperactivation 
processes of free radical oxidation is observed 
in the 1st day, reaching a peak in 7–14th days of 
post­traumatic period in blood, tissues, liver, 
heart and lungs if compared to the control 
group that causes multiple organ failure.

The future prospects of the research are 
investigation of oxidant system dynamics after 
traumatic injuries of chest and hips and estab­
lishment of antioxidant­prooxidant disbalance.

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Received: 2016-12-10

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