Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 241248

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Spectrochimica Acta Part A: Molecular and

Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa

Determination of aluminium induced metabolic changes in mice liver: A

Fourier transform infrared spectroscopy study
S. Sivakumar a,, J. Sivasubramanian a, Chandra Prasad Khatiwada a, J. Manivannan b, B. Raja b
a
b

Department of Physics, Annamalai University, Annamalai Nagar 608 002, Tamil Nadu, India
Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar 608 002, Tamil Nadu, India

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 FTIR spectra determine the

aluminium toxicity induced

metabolic changes in liver tissue.
 FTIR results revealed the quantitative
and qualitative alterations of major
biochemical constituents.
 Results of the FTIR study were found
to be in agreement with biochemical
studies.

a r t i c l e

i n f o

Article history:
Received 19 November 2012
Received in revised form 14 February 2013
Accepted 4 March 2013
Available online 21 March 2013
Keywords:
FTIR
Liver
Aluminium
ICP-OES

a b s t r a c t
In this study, we made a new approach to evaluate aluminium induced metabolic changes in liver tissue of
mice using Fourier transform infrared spectroscopy analysis taking one step further in correlation with
strong biochemical evidence. This nding reveals the alterations on the major biochemical constituents,
such as lipids, proteins, nucleic acids and glycogen of the liver tissues of mice. The peak area value of amide
A signicantly decrease from 288.278 3.121 to 189.872 2.012 between control and aluminium treated
liver tissue respectively. Amide I and amide II peak area value also decrease from 40.749 2.052 to
21.170 1.311 and 13.167 1.441 to 8.953 0.548 in aluminium treated liver tissue respectively. This
result suggests an alteration in the protein prole. The absence of olenic@CH stretching band and C@O
stretching of triglycerides in aluminium treated liver suggests an altered lipid levels due to aluminium
exposure. Signicant shift in the peak position of glycogen may be the interruption of aluminium in the
calcium metabolism and the reduced level of calcium. The overall ndings exhibit that the liver metabolic
program is altered through increasing the structural modication in proteins, triglycerides and quantitative alteration in proteins, lipids, and glycogen. All the above mentioned modications were protected in
desferrioxamine treated mice. Histopathological results also revealed impairment of aluminium induced
alterations in liver tissue. The results of the FTIR study were found to be in agreement with biochemical
studies and which demonstrate FTIR can be used successfully to indicate the molecular level changes.
2013 Elsevier B.V. All rights reserved.

Introduction
Aluminium (Al) is the third most prevalent element and the
most abundant metal in the earths crust, i.e., approximately 8%
Corresponding author. Tel.: +91 9865043811.
E-mail address: girihari777@yahoo.com (S. Sivakumar).
1386-1425/$ - see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.03.056

of total mineral components [1]. Al is widely distributed in the

environment and extensively used in daily life resulting easy exposure to human beings [2]. From the environment it gets access to
the human body via the gastrointestinal and the respiratory tracts.
Aluminium is known to disrupt the pro-oxidant/antioxidant balance of tissues, which leads to various biochemical and physiological dysfunction [3]. Some studies reported that Al accumulates in

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liver and kidney [4,5]. Aluminium exposure also results in the production of free radicals [6]. ROS are highly toxic and can react with
lipids, proteins and nucleic acids, and lead to cell death via apoptosis or necrosis [7]. Al accumulation in liver induces oxidative stress
with an increase in thiobarbituric acid-reactive substances (TBARS)
and to a decrease in the levels of antioxidant defences, including
reduced glutathione (GSH) and the antioxidant enzymes, catalase
(CAT) and glutathione peroxidase (GSH-Px) [8]. Al exposure can result in Al accumulation in the liver leading to a disturbance of lipid
metabolism and an elevation of serum cholesterol [9]. Aluminium
induces cholestasis associated with multiple alterations in hepatocellular transporters involved in bile secretory function, like Mrp2
[10]. Al exposure has an adverse effect on essential elements in
humans, with subsequent impact on the cellular enzymatic and
metabolic processes [11]. Inductively Couple Plasma Optical Emission Spectroscopy (ICP-OES) is an important technique to study the
trace elements at molecular level in various biological samples. It is
particularly effective in this context due to its high sensitivity for
detecting the major trace elements [12].
For the last several decades the most widely used chelator in
the treatment of aluminium intoxication has been desferrioxamine
(DFO) [13]. The treatment commonly used in aluminium disorders
is desferrioxamine (DFO), which is a chelator with great capacity to
decrease Al body burden by increase its excretion in the urine. This
compound is usually employed in iron accumulation and since
there are chemical and physical similarities among aluminium
and iron (charge, ionic radius and protein binding) it has been used
in cases of aluminium accumulation [14].
Fourier transform infrared spectroscopy (FTIR) is one of the
most important metabolomic tools showing the molecular changes
that occur during a pathological condition. IR spectroscopy can distinguish differences in the characteristics of diverse molecules by
probing chemical bond vibrations and use these molecular and
sub-molecular proles to dene and differentiate diseased and
healthy tissues [15]. The frequency shifts shows the molecular
alteration of macromolecules such as protein, lipid, carbohydrate,
and nucleic acid can be considered for analysis [16]. As such infrared spectroscopy is a very valuable tool for biochemical investigations [17]. Previously, 17-b Estradiol induced compositional,
structural and functional changes in rainbow trout liver has been
investigated using FTIR spectroscopy [18]. Therefore, the main
objective of this study was to use this molecular ngerprinting approach to investigate the effects of aluminium on metabolic alterations in liver tissues of mice.

Treatment schedule
Mice were randomly allocated in three different groups. Each
group contains 12 animals. Group I served as control and were
fed on standard animal chow and water ad libitum. Animals in
groups II & III were administered aluminium in the form of aluminium chloride (100 mg/kg b.wt./day) orally for a period of 12 weeks.
Following to 12 weeks of AlCl3 administration, group III mice were
treated with DFO (s.c.) at the dose of 0.89 mmol/kg for ve consecutive days. Aluminium chloride was dissolved in normal drinking
water and was given by an oral gavage. Twenty-four hours after
the last administration of the chelator, mice were anesthetized
with diethyl ether and killed. Six liver samples from each group
were immediately homogenized and used for the various biochemical estimations. For FTIR analysis six whole liver samples of all
three groups were freeze dried and homogenized with liquid nitrogen using agate mortar and pestle. Samples were stored under
80 C until used. Even though samples in dried form reduced
the interference of water, it has some limitations. The bands become much broader in the dry sample, leading to the disappearance of the ne structure observed in the wet sample and the
dry sample method is not suitable for characterize the living cells.
FTIR sample preparation
For FTIR analysis, the samples were mixed with KBr at ratio of
1:100. The mixture was then subjected to a pressure of 1100 kg/
cm2 to produce KBr pellets for use in FTIR Spectrometer. Pellets
of the same thickness were equipped by taking the same quantity
of sample and applying the same pressure. Therefore, in the current study, it was possible to directly relate the intensities of the
absorption bands to the quantity of the corresponding functional
groups.
FTIR spectra and data analysis
FTIR spectra of the region 4000400 cm 1 were recorded at the
temperature of 25 1 C on a Nicolet-Avatar-360 FTIR Spectrometer equipped with an air-cooled DTGS (deuterated triglycine
sulfate) and purged with nitrogen. Each sample was scanned with
three different pellets under identical conditions. These replicates
were averaged and then used. The spectra were analyzed using
ORIGIN 6.0 software (OriginLab Corporation, Massachusetts, USA).
ICP-OES analysis

Materials and methods

Animals
Male Swiss albino mice, weighing 25 2 g, were procured from
the central animal house, Department of Experimental Medicine,
Rajah Muthiah Medical College, Annamalai University, and were
maintained in an air-conditioned room (25 1 C) with a 12 h
light/12 h dark cycle. Feed and water were provided ad libitum
to all the animals. The study was approved by the Institutional
Animal Ethics Committee of Rajah Muthiah Medical College and
Hospital (Reg. No. 160/1999/CPCSEA, Proposal number: 851),
Annamalai University, Annamalai Nagar.
Test chemicals
Desferrioxamine (Desferal) was purchased from Novartis and
all other chemicals used in this study were of highest analytical
grade obtained from Sisco Research Laboratories and Himedia,
Mumbai, India.

Chronic aluminium exposure on mineral metabolism and the

level of essential elements was determined by using Inductively
Coupled Plasma Optical Emission Spectrometry (ICP-OES, Perkin
Elmer Optima 2100 DV model) available at CAS in Marine Biology,
Annamalai University, by standard digestion method [19]. Blank
solution was prepared for the background correction. All the analyses were carried out in six replications and the average values
have been reported.
Biochemical analysis
Estimation of lipid peroxidation, protein level and enzymic
antioxidants
The concentration of thiobarbituric acid reactive substances
(TBARS) was estimated by the method of Niehius and Samuelsson
[20]. Protein levels of liver of mice were estimated by the method
of Lowry et al. [21]. Lipid hydroperoxides (LHPs) was estimated by
the method of Jiang et al. [22]based on the oxidation of ferrous ion
(Fe2+) under acidic conditions in the presence of xylenol orange

S. Sivakumar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 241248

that leads to the formation of a chromophore with an absorbance

maximum at 560 nm. The activities of enzymatic antioxidants
super-oxide dismutase (SOD) catalase (CAT) and glutathione peroxidase (GPx) were assayed by the methods of Kakkar [23], Sinha
[24] and Rotruck [25] respectively.
Activities of serum hepatic marker enzymes
Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were estimated using the method of Reitman
and Franke [26]. Alkaline phosphatase was estimated using the
method of Kind and King [27]. Serum bilirubin was estimated by
the method of Malloy and Evelyn [28].
Estimation of non-enzymic antioxidant
GSH was estimated by the method of Ellman [29]. This method
was based on the formation of 2-nitro-5-thiobenzoic acid when
dithionitro benzoic acid was added to compounds containing sulfhydryl groups. The yellow colour developed was read at 412 nm.
The level of vitamin C was assessed by the method of Roe and
Kuether [30]. The ascorbic acid was converted to dehydroascorbic
acid and then coupled with 2,4-dinitrophenylydrazine (DNPH) in
the presence of thiourea, a mild reducing agent. The coupled dinitrophenylhydrazine was converted into a red coloured compound
when treated with sulfuric acid and read at 520 nm. Vitamin E
was estimated by the method of Baker et al. [31]. The method involves the reduction of ferric ions to ferrous ions by a-tocopherol
and the formation of a red colored complex with 2,2-dipyridyl.
Absorbance of the chromophore was measured at 520 nm.
Histological examination of liver tissues
Excised liver samples were cleared from blood and immediately
xed in a neutral buffered solution of 10% formalin for 24 h. 5 lmthick tissues sectioned from the liver of each animal were prepared
for parafn-embedded samples process. The liver tissue sections
were stained with Hematoxylin and Eosin (H&E) for light microscopic examination for the evidence of aluminium induced
changes. The cross-sectional area (CSA) of liver was evaluated from
photographs of whole tissue sections taken at 100 magnication
and scanned, digitized and analyzed by computer, using the Adobe
Photoshop Imaging Program (Adobe System Incorporation).
Statistical analysis
Values are given as means S.D. for six mice in each group. Data
were analyzed by one-way analysis of variance followed by
Duncans Multiple Range Test (DMRT) using SPSS version 11.5
(SPSS, Chicago, IL). The limit of statistical signicance was set at
P < 0.05.
Results
The FTIR spectroscopy monitors the vibration modes of functional groups present in proteins, lipids, polysaccharides, and
nucleic acids. FTIR allows one to get chemical information on molecules within the sample. Variations in the positions, widths, and
strengths of these modes with composition and structure allow
molecular species to be uniquely identied, including the functional groups of interest in cells [32]. Shifts in peak positions,
changes in bandwidths, intensities, and band area values of the
infrared bands are used to obtain valuable structural and functional information about the system of interest and the intensity
and/or more accurately the area of the absorption bands is directly
related to the concentration of the molecules [33]. The spectra of
all groups are shown in Fig. 1. As could be seen from the gures

243

displayed, considerable changes are observed in the intensities of

the bands. The tentative vibrational frequency assignments of
absorption spectra are presented in Table 1. Fig. 2 shows the detailed spectral analyses of three distinct frequency ranges. 3800
2700 cm 1 (A); 18001400 cm 1 (B); and 1400400 cm 1 (C),
and the observations made are described below. The frequency value changes of important functional groups and band area values
are provided in Tables 2 and 3 respectively.
Infrared spectra from control, aluminium intoxicated and DFO treated
tissue
The wavenumber of the region shown in Fig. 2A mainly consists
of amide A vibrations of proteins. As shown in Table 1, the bands in
this region arise from NAH and OAH stretching modes of proteins,
polysaccharides and intermolecular hydrogen bonding [18]. Amide
A appeared at 3419 cm 1 in control and 3417 cm 1 in aluminium
treated liver tissue. There is no signicant change in wavenumber
between control and aluminium intoxicated group. But the peak
area value of amide A band decreases (34.13%) signicantly from
288.278 3.121 to 189.872 2.012 between control and aluminium treated liver tissue respectively. Treatment with DFO brings
back the changes to fairly near (231.449 3.448) the control band
area value. There is a (21.89%) signicant increase in DFO treated
group when compared with aluminium intoxicated group. The olenic acid band which is due to CAH stretching mode of the HC@CH
groups, can be used as a measure of unsaturation in the acyl chains
act as indicator of unsaturated lipids [18]. In this study olenic@CH
stretching band was not observed in aluminium treated liver tissue. The peak observed at 2958 cm 1 was assigned to asymmetric
stretching vibrations of methyl group. This band mainly monitors
the lipids in the biological system [34]. The CH2 asymmetric band
at 2923 cm 1 and CH2 symmetric band at 2854 cm 1 is due to
stretching of lipids, the frequency number of these regions was
not changed signicantly (P < 0.05) in all the groups.
The wavenumber region shown in Fig. 2B, is mainly due to proteins, with some absorbance from lipids. The peak observed at
1740 cm 1 indicates C@O stretching of triglycerides in liver tissues
[35] and this frequency value is not observed in aluminium treated
group. This region mainly originates from amide I and amide II proteins. In the present study, there is no signicant change in wavenumber between control and aluminium intoxicated group. But
the peak area value of amide I band decreases (48.04%) signicantly from 40.749 2.052 to 21.170 1.311 between control
and aluminium treated liver tissue respectively. In the DFO treated
group administration of DFO considerably bring back the protein
level (35.813 1.965). The administration of chelating agent DFO
69.16% improves the protein level when compare with aluminium
intoxicated liver tissue. This indicates the signicant protection of
amide linkage from aluminium induced damages in DFO treated
group. The amide II absorption arises from amide NAH bending
vibration coupled with CAN stretching vibration mode of the polypeptide and protein back bone. In the present study amide II was
observed at 1542 cm 1 in the control liver tissue and did not exhibit any signicant change in wavenumber between all three
groups. The peak area of this band (32%) decreased in aluminium
intoxicated group (8.953 0.548) and this reduction indicates the
decreases of quantity of amide II protein. The amino acid side chain
from peptides and proteins at 1459 cm 1 is associated with the
CH2 bending vibration of lipids and proteins. The intensity of this
band also decreased from 1.214 0.453 to 1.150 0.068. The
absorption band at 1398 cm 1 is assigned as COO symmetric
stretching vibration of amino acid side chains and fatty acids. There
is no signicant difference between the wavenumber of all groups.
In Fig. 2C, the absorptions are primarily due to the existence of
polysaccharides, nucleic acid, and minerals. The strong bands at

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Fig. 1. FT-IR spectra of the control, aluminium intoxicated and DFO treated liver tissue of mice.

Table 1
FT-IR vibrational assignment for control, aluminium intoxicated and DFO treated liver tissue of mice.
Wavenumber (cm

Denition of the spectral assignment

Control

Aluminium
treated

Aluminium + DFO
treated

3419

3417

3415

3012
2958
2923
2854
1740
1654
1542
1457
1398
1234
1163
1082
1048
970

Not observed
2959
2925
2854
Not observed
1654
1542
1456
1396
1237
1159
1083
1045
968

3016
2959
2926
2853
Not observed
1652
1543
1456
1396
1236
1164
1083
1049
971

Amide A: mainly NAH stretching of proteins with the little contribution from OAH stretching of
polysaccharides.
olenic@CH stretching, unsaturated fatty acids.
CH3 asymmetric stretch: mainly lipids
CH2 asymmetric stretch: mainly lipids
CH2 symmetric stretching lipids (fatty acids)
Ester C@O stretch: triglycerides, cholesterol esters
Amide I: C@O stretching of proteins
Amide II: NAH bending and CAN stretching of proteins
CH2 Bending: mainly lipids, with the little contribution from proteins
COO symmetric stretch: fatty acids and amino acids
PO2 asymmetric stretch: mainly nucleic acids with the little contribution from phospholipids
COAOAC asymmetric stretching: glycogen and nucleic acids
PO2 symmetric stretch: nucleic acids and phospholipids
CAO stretching: polysaccharides
CAN+AC symmetric stretch: nucleic acids

1234 and 1082 cm 1 are mainly due to asymmetric and symmetric

stretching modes, respectively, of phosphodiester groups in nucleic
acids rather than in phospholipids [34]. As shown in Fig. 2C, and
Table 3, the aluminium treated liver tissue spectrum displayed a
decrease in the area value of stretching bands with respect to those
of control tissue. In addition, the phosphate asymmetric stretching
band shifted to higher frequency value. The wavenumber of 1156
and 1073 cm 1 are the indicators of glycogen which is due to the
stretching mode of COAOAC groups present in glycogen, a major
molecule of liver metabolism [36]. In the present study glycogen
was observed at 1163 cm 1 in the control liver tissue and the
wavenumber was appeared at 1159 cm 1 in aluminium treated
liver tissue of mice. The wavenumber of DFO treated group appeared at 1164 cm 1. This wavenumber signicantly shifted to
lower wavenumber in aluminium intoxicated liver tissue and the
administration of DFO bring back the wavenumber nearer to the
control group. The band at 1048 cm 1 is mainly assigned to CAO
stretching vibrations in polysaccharides [37]. As seen from Table
1 the wavenumber shifted to lower wavenumber in aluminium
intoxicated liver tissues. This signicant shift indicates a change

in the polysaccharides metabolism and the peak area value also decreased in aluminium intoxicated group. The band centered at
970 cm 1 is generally assigned to symmetric stretching mode of
dianionic phosphate monoester of cellular nucleic acids, especially
for DNA.
ICP-OES analysis
The elements such as Ca, Fe, Cu, Mn and Zn are essential elements
because of their important role in biological systems. Aluminium
may interfere with various metabolic processes, in which Ca2+,
Mg2+, Fe3+ and Fe2+ (gastrointestinal absorption) are involved [38].
ICP-OES results show the interaction of aluminium with essential
elements in Table 4. The choice of those elements was based in previous studies which showed potential relationship between aluminium and the above mentioned elements [39]. In the present study
the intoxicated mice showed a reduction in the level of calcium, further the administration of DFO also not shown any increment in the
level of calcium. Tissue concentration of other elements level did not
show any signicant difference except Calcium.

S. Sivakumar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 241248

245

Fig. 2. Selected wavenumber regions of control, aluminium intoxicated and DFO treated liver tissue of mice in the range of 38002700 cm1 (A); 18001400 cm1 (B); 1400
400 cm1 (C).

Table 2
Wave number alterations of major macromolecular groups.
Molecular vibration

Control

Aluminium treated

Aluminium + DFO treated

NAH stretching (proteins)

Olenic@CH (lipid unsaturation)
Carbonyl C@O stretch: lipids
PO2 asym. stretch nucleic acids
CAO stretching: polysaccharides
Glycogen

3419.14 2.14
3012.17 0.32a
1740.10 0.12a
1234.22 1.08a
1048.31 0.21a
1163.64 0.19a

3417.19 2.11
Not observed
Not observed
1237.13 0.52
1045.13 0.12
1159.29 0.31

3415.21 2.18
3016.12 0.09a
Not observed
1236.38 0.16
1049.16 0.19a
1164.12 0.22a

Values are expressed as mean S.D. for six mice in each group.
a
The frequency values are shifted signicantly compared with aluminium treated (P < 0.05) animal Liver spectra.

Biochemical and histopathological examination

Table 5 shows the effects of DFO on the levels of thiobarbituric
acid reactive substances (TBARS) and on the activities of superoxide dismutase, catalase and glutathione peroxidase in liver tissues
of aluminium intoxicated mice. TBARS is an end product of lipid
peroxidation. Aluminium might increase the level of TBARS in
the tissues of experimental animal [40]. In the present study aluminium intoxicated mice showed a signicant increase in the levels of thiobarbituric acid reactive substances and the
administration of DFO reduced the levels of thiobarbituric acid
reactive substances. The activities of superoxide dismutase, catalase and glutathione peroxidase decreased signicantly in intoxi-

cated mice and the administration of DFO signicantly increased

these enzymatic antioxidants.
In the clinical plasma examination, the activities of AST and ALT
in plasma represent biomarkers for liver functions. The activities of
AST and ALT in the plasma of rats were signicantly elevated by
aluminium, indicating aluminium related injury to the liver [41].
Usually, the extent of hepatic damage is assessed by the increased
level of cytosolic enzymes such as ALT, AST, ALP and LDH [42]. In
the present study, the changes in the activities of serum hepatic
markers in control and aluminium treated groups are given in Table 6. The activities of AST, ALT, ALP, and the levels of bilirubin
were signicantly (P < 0.05) increased in intoxicated mice group,
further the administration of desferrioxamine showed signicantly

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Table 3
Band area variations of macromolecular groups from quantitative analysis of spectra.

Table 7
Levels of Non-enzymic antioxidants in control and experimental mice.

Wavenumber
(cm 1)

Control

Aluminium
treated

Aluminium + DFO
treated

Groups

GSH (mg/100
g tissue)

Vitamin C
(lg/mg protein)

Vitamin E
(lg/mg protein)

3419
3012
2958
2923
2854
1740
1654
1542
1457
1398
1234
1163
1082
1047
970

288.278 3.121a
0.213 0.615a
0.713 0.142a
4.563 0.579a
1.355 0.462a
0.360 0.082a
40.749 2.052a
13.167 1.441a
2.393 0.397a
4.826 1.273a
5.690 1.181a
0.312 0.038a
1.087 0.237a
0.408 0.035a
0.091 0.008a

189.872 2.012

0.438 0.235
2.007 1.470
0.941 0.615

21.170 1.311
8.953 0.548
1.056 0.085
1.354 0.179
2.157 0.394
0.214 0.725
0.684 0.647
0.173 0.065
0.047 0.036

231.449 3.448a
0.353 0.067a
0.575 0.064a
2.315 1.147
0.802 0.624

35.813 1.965a
10.984 0.704a
1.468 0.009a
3.609 0.581a
3.515 0.168a
0.256 0.048
0.643 0.241
0.080 0.009
0.096 0.021a

Control
Al
Al + DFO

93.50 5.26
60.82 5.60a
85.63 6.27b

1.86 0.16
0.74 0.92a
1.32 0.40b

4.84 0.72
2.31 0.25a
3.91 0.17b

Values are expressed as mean S.D. for six mice in each group.
a
The band area values are shifted signicantly compared with aluminium treated (P < 0.05) animal Liver spectra.

Table 4
concentrations of essential elements in liver tissue of control, aluminium intoxicated
and DFO treated mice (lg/g tissue).
Groups

Calcium

Magnesium

Zinc

Copper

Iron

Control
Al
Al + DFO

92 5.7
76 8.3a
78 9.7a

194 34.5
190 29.4
188 22.1

26 3.6
25 4.4
24 3.7

4.8 1.1
5.2 0.8
5.5 0.9

208 31.5
216 36.9
222 43.4

Values are expressed as mean S.D. for six mice in each group.
a
Signicant as compared to control (P < 0.05).

Table 5
Effect of DFO on protein lipid peroxidation product and enzymatic antioxidants in
aluminium intoxicated mice.
Parameters

Control

Al intoxicated

Al + DFO

Protein level (mg/100 mg)

TBARS (nmol/mg protein)
LHP (nmol/mg protein)
SOD (U/mg of protein)
CAT (l mol of H2O2 utilized/
min/mg protein)
Gpx (lg of GSH utilized
min/mg protein)

35.13 0.29
1.95 0.09
0.95 0.12
6.45 0.31
52.76 5.42

20.52 0.22a
4.62 0.48a
3.92 0.25a
3.98 0.37a
28.51 4.31a

26.61 0.35b
2.87 0.71b
1.86 0.15b
4.83 0.42b
41.74 6.33b

11.54 1.61

6.91 1.25a

9.62 0.95b

Values are expressed as mean S.D. for six mice in each group.
a
Signicant as compared to control (P < 0.05).
b
Signicant as compared to Al intoxicated (P < 0.05).

Table 6
Effect of DFO on changes in the serum AST, ALT and ALP of aluminium intoxicated
mice.
Groups

AST (IU/L)

ALP (IU/L)

ALT (IU/L)

Bilirubin (mg/
dl)

Control
Al
Al + DFO

73.50 5.26
109.10 8.10a
89.63 6.27b

70.36 5.61
101.04 8.50a
83.70 6.30b

32.88 2.70
61.91 4.60a
41.92 2.78b

0.57 0.03
1.20 0.90a
0.83 0.11b

Values are expressed as mean S.D. for six mice in each group.
a
Signicant as compared to control (P < 0.05).
b
Signicant as compared to Al intoxicated (P < 0.05).

(P < 0.05) decreased activities of serum AST, ALT, ALP, and bilirubin.
The levels of nonenzymic antioxidants (vitamin C, vitamin E, and
GSH) in the control and experimental mice liver are presented in
Table 7. GSH is involved in the mechanism of detoxication scav-

Values are expressed as mean S.D. for six mice in each group.
a
Signicant as compared to control (P < 0.05).
b
Signicant as compared to Al intoxicated (P < 0.05).

enging free radicals in rat liver [43]. Vitamins C and E were studied
to minimize Al toxicity [44]. The levels of vitamins C and E and GSH
were signicantly (P < 0.05) reduced in Al-treated mice when compared to control mice. Administration of DFO signicantly
(P < 0.05) increased the levels of nonenzymic antioxidants in Aladministered mice liver.
Fig. 3AC shows the control, aluminium intoxicated and effect
of DFO on the histology of liver tissue in aluminium intoxicated
mice, respectively. Control liver shows normal hepatic parenchyma
(Fig. 3A). Fig. 3B shows the histopathological nding of aluminium
intoxicated liver with diffused fatty change. In Fig. 3C, Al + DFO
administrated liver shows proliferative hepatocytes.
Discussion
Al exposure has a most important, damaging impact on hepatocellular integrity and function. The liver has facilitated specic
transport systems for Al uptake, and similar to other organs that
accumulate Al, it is not surprising that the liver is a target for Al
toxicity [9]. FTIR spectroscopy could be a convenient tool for monitoring metabolic changes in liver tissue after in vivo exposure to
chemical drugs, and that it might be used in conjunction with biochemical and physiological data [45]. In this study, we examined
the effects of DFO on aluminium induced changes in mice liver tissue at molecular level using FT-IR spectroscopy. The FTIR analysis
explores the vibration of functional groups present in macromolecules and shows the molecular structural changes of the molecules
through shifts in wavenumbers. The signal intensity, or more accurately, the band area gives information about the concentration of
related functional groups [33]. So in this study, the decreased
intensity of amide A band in the aluminium intoxicated liver tissue
reects the decreased quantity of protein. This decreased quantity
indicates the damage of amide linkage in aluminium intoxicated liver tissue. As can be seen from Table 5, there was an increment in
the level of thiobarbituric acid reactive substances and lipid hydroperoxides. Oxidative stress occurs when ROS levels exceed the
antioxidant capacity of a cell. Al generates reactive oxygen species,
resulting in oxidative deterioration of lipids, proteins and DNA.
Makrides [46] suggested that free radical damage can cause a
reduction in protein synthesis. The decreased antioxidant activities
could bedue to the induction of lipid peroxidation [47]. Tables 5
and 6 show the depletion of enzymic and non enzymic antioxidants level in aluminium intoxicated liver tissue. So the reduction
of protein quantity may be the imbalance between oxidants and
antioxidants. DFO is also a useful therapeutic tool in blocking
ROS production [48]. In this study the administration of DFO bring
back the protein level near to control value.
ROS may attack any type of molecules, but their main target appears to be polyunsaturated fatty acids (PUFAs), the precursors of
lipid peroxide formation [49]. It is known that unsaturated lipids
are more prone to lipid peroxidation [50]. Unsaturated lipids are
highly vulnerable to oxidative attack because of their double bond
content. FT-IR is usefulness in monitoring unsaturated lipid content by utilizing the olenic @CH stretching mode 3012 cm 1

S. Sivakumar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 241248

247

Fig. 3. Representative photomicrograph of histological changes in liver. (A) Control, normal hepatic parenchyma. (B) Aluminium intoxicated, there was diffused fatty change.
(C) Al + DFO, shows proliferative hepatocytes.

Table 8
Band area ratio for selected bands of liver tissues of mice.
Band area ratio

Control

Aluminium treated

Aluminium
+ DFO treated

I2959/I2854
I1082/I1542
I2923/I2958

0.526 0.013
0.082 0.004
6.399 0.166

0.465 0.016*
0.076 0.003
4.582 0.142

0.716 0.020
0.058 0.003
4.026 0.138

Values are shown as mean standard error; the degree of signicance was denoted
as:
P < 0.001.
*
P < 0.01.

P < 0.05 for comparisons of aluminium intoxicated to control group.

[33]. The unsaturated olenic C@CAH stretching vibration, which

has a unique vibrational frequency of 3012 cm 1 and is well separated and distinguishable from the saturated aliphatic peaks [51].
In the present study the olenic C@CAH peak almost disappear
in the aluminium intoxicated liver tissue. This may be due to vulnerable attack of aluminium induced lipid peroxidation. This result
is supported by Carbonyl C@O stretching vibration of lipids at
1733 cm 1. Further the administration of DFO blocked the ROS
production and increased the level of polyunsaturated fatty acids.
Ester C@O stretch of triglycerides diminished in the intoxicated liver indicates a difference in packing of ester groups within the
tissue.
The peak area of the CH3 asymmetric stretching vibration at
2957 cm 1 decreased as a result of aluminium intoxication. This
decrease indicates a change in the composition of the acyl chains
[52]. CH2 asymmetric and CH2 symmetric stretching of lipids
shows no shift in frequency. The area of the symmetric CH2
stretching band was found to be signicantly decreased in the aluminium treated tissues. This result suggested decreased proportion
of the CH2 groups in the aluminium treated liver tissues. The ratios
of the absorption intensities of selected bands of liver tissues are
given in Table 8. The mean ratio (I2959/I2854) of the intensity of
absorption of the methyl band and the methylene band for control,
aluminium intoxicated and DFO treated liver tissues are, respectively, 0.526 0.013, 0.465 0.016 and 0.716 0.020 respectively.
The decreased ratio indicates a decrease in the number of methyl
group compared to methylene group in the aluminium intoxicated
liver tissues. The increased ratio further indicates the contribution
for high number of protein bers [16]. In this study the decreased
ratio in aluminium intoxicated liver tissue may specify the contribution of low number of protein bers. Further the DFO treated
group shows the high number of protein bers. The ratios of peak
intensities of the bands (I1082/I1542) are found to be 0.082 0.004,
0.076 0.003 and 0.058 0.003 for the control, aluminium intoxicated and DFO treated liver tissues, respectively. These intensity
variations indicate decrease in the glycoprotein content in the aluminium intoxicated liver tissues. The ratios between the areas of
absorbance bands I2923/I2958 give information on molecular
changes occurring in the lipids. In the present study, these ratios

are (28.39%) lower from 6.399 0.166 to 4.582 0.142 between

control and aluminium intoxicated tissues, respectively. This may
be caused by different content of lipids with longer aliphatic chains
[53].
There is no shift in the amides I and II bands. As it is clearly seen
in Table 3, the area values of the amides I and II, consequently the
amount of proteins, decreased in the aluminium intoxicated liver
tissues. This decrement was consistent with the decrease in amide
A band due to proteins. This quantity depletion may be due to the
increment of peroxidation and the reduction of antioxidants. This
result is supported by biochemical studies. The reduction of protein quantity may be due to signicant imbalance between peroxidation and antioxidants. This reduced intensities of the amide I
and II bands can be interpreted as the result of modication of
the protein synthesis and the protein structure due to aluminium
intoxication. Further the administration of DFO inhibits the level
of peroxidation and takes back the level of protein nearer to the
control value. The decline in the intensity of asymmetric and symmetric phosphate stretching band implies decrement in the relative content of the nucleic acids.
Aluminium shows high afnity to interact with ATP molecule
[54]. The increased level of ALT in serum of mice that were treated
with aluminium may be due to the cellular energy depletion, interference of aluminium ions with phosphate and ATP metabolism, or
changes in cell membrane integrity [55]. In the present study the
level of ALT signicantly increased in the aluminium intoxicated liver tissue of mice. This may indicate the interaction of aluminium
with ATP metabolism. ATP is important in carbohydrate metabolism and also interacts with aluminium. This mechanism may explain the carbohydrate metabolism is disturbed by aluminium.
Solheim and Fromm reported the inhibition of hexokinase, a key
enzyme in glucose metabolism, by aluminium leads to the decreased glucose uptake and consumption by the cells [56]. So in
the present study shift in the peak position and the decreased level
of glycogen in aluminium intoxicated liver tissue may indicate the
interaction of aluminium with ATP metabolism and inhibition of
hexokinase. In the DFO administrated group the peak position of
glycogen appeared nearer to the control value.
El Sayed et al. identied the change in the permeability of the
mitochondrial membrane due to aluminium exposure in the liver
tissue of mice [57]. An increase in mitochondrial membrane permeability contributes to changes in the redox status of the mitochondrial thiol groups which in turn may affect the cellular free
calcium levels [58]. Several data suggest that Al3+ can interact with
Ca2+ binding sites probably due to their similar atomic radii and
henceforth may play a part in disruption of calcium homeostasis
[59] As can be seen in Table 4 the level of Calcium decreased in aluminium treated liver tissue. This may specify the interruption of
aluminium in the calcium binding sites and change in the permeability of the mitochondrial membrane due to aluminium exposure. Hashimoto et al. suggested that Ca2+calmodulin may be
involved as one of the important factors causing this glycogen synthase inactivation [60]. So, in this study interruption of aluminium

248

S. Sivakumar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 241248

in the calcium metabolism may also be the reason for the changes
in the glycogen peak position.
At the molecular level, any effects of Al would be most likely to
involve DNA damage. Al does have a genotoxic prole and has been
shown to bind to DNA [61]. The band centered at 970 cm 1 is generally assigned to cellular nucleic acids, especially for DNA. The
stronger nucleic acid band implied an increase in the relative content of the nucleic acids in the treated tissues [62]. In this study the
peak area of nucleic acid decreased in aluminium intoxicated liver
tissue of mice. This indicates the decline in the relative content of
the nucleic acids and the deleterious effect of aluminium in DNA.
Conclusion
The present studies demonstrate FTIR spectra reveals signicant
changes in absorbance intensities between the control and aluminium intoxicated liver tissues. Further, the administration of DFO
considerably recover the levels of biochemical constituents. The
experimental results suggest that quantity of protein signicantly
decreased due to the imbalance between oxidants and antioxidants. Quantity of unsaturated fatty acid decreased may indicate
the increases of lipid peroxidation. Interaction of aluminium with
ATP metabolism and calcium homeostasis has been articulate
through the Shift in the peak position and the decreased level of
glycogen in aluminium intoxicated liver tissue. In addition FTIR results were found to be in agreement with biochemical studies. Finally the present study shows that the FTIR spectroscopy is likely
to be a very informative technique to determine the aluminium
toxicity induced changes in liver tissue at molecular level.
Acknowledgment
The nancial support to Mr. J. Sivasubramanian as project fellow from the University Grants Commission, New Delhi, India is
gratefully acknowledged.
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