Anal. Methods Environ. Chem. J. 4 (4) (2021) 78-91
Research Article, Issue 4
Analytical Methods in Environmental Chemistry Journal
Journal home page: www.amecj.com/ir
AMECJ
Determination of manganese in rat blood samples based
on ionic liquid-liquid extraction and chelation therapy for
evaluation of manganese toxicity in rats
S. Jamilaldin Fatemia,*, Tayyebeh Zandevakilia, Fatemeh Khajoee Nejada, Marziyeh
Iranmaneshb and Mohammad Faghihi Zarandic
a Department of Chemistry, Shahid Bahonar University of Kerman, Kerman, Iran
b Department of Chemistry, Kerman Branch, Islamic Azad University, Kerman, Iran
c Department of Foreign Languages, Shahid Bahonar University of Kerman, Kerman, Iran
ABSTRACT
In this study, the manganese ions were extracted in the blood of rats based
on desferrioxamine (DFO), deferasirox (DFX) and deferiprone (DFP)
as chelators (ligands) by ionic liquid-liquid phase extraction method
(ILLEM) before being determined by F-AAS. Also, the toxic effects of
manganese on blood serum and hematology parameters such as: RBC,
WBC, HGB, PLT and HCT were investigated. Male Wistar rats were
randomly divided into control and toxic groups. Manganese chloride
was administrated orally in low and high doses. Orally (deferasirox and
deferiprone) or intraperitoneally (desferrioxamine) for 2 weeks. Results
of hematology parameters and concentration of this metal ion in serum
compared to the control group. By procedure, manganese was chelated
with ligands in the blood of rats and then the hydrophobic ionic liquid
(IL, [HMIM][PF6]) was added to blood samples. After shaking and
centrifuging, the upper liquid phase was separated by an auto-sampler
and manganese loaded in a mixture of IL/ligand was settled down in the
bottom of the conical tube. The manganese ions were back-extracted
from IL phase and the remained acid solution was determined with
F-AAS. The linear range, LOD and enrichment factor for 10 mL of blood
-1-1 and 19.92, respectively.
By chelation therapy extra manganese ions were removed from human
serum and the normal hematology parameters were achieved.
Keywords:
Chelators,
Red blood cells,
Toxicity,
Ionic liquid-liquid extraction,
Flame atomic absorption spectrometry
ARTICLE INFO:
Received 22 Aug 2021
Revised form 28 Oct 2021
Accepted 23 Nov 2021
Available online 28 Dec 2021
*Corresponding Author: S. Jamilaldin Fatemi
Email: fatemijam@uk.ac.ir
https://doi.org/10.24200/amecj.v4.i04.160
------------------------
1. Introduction
Trace elements like manganese are essential for
normal development and body function across the
life span, and are widely distributed in the tissues.
Manganese is required for normal amino acid,
lipid, protein, and carbohydrate metabolisms.
Enzyme families that are Mn-dependent include
oxidoreductases, transferases, hydrolases, lyases,
causes improper organism function, but toxicity
results if Mn is present in excessive amounts
[1,2]. Concentrations of metal in erythrocytes
may be a good indicator of tissue accumulation
since red blood cells account for about 60–80% of
the metal found in whole blood [3-5]. Manganese
leaving the enterocyte and entering the circulation
bound to transferrin, may bind to transferrin
receptors on erythroid cells. The erythroid cell
79
Manganese separation with ILLEM and toxicity evaluation S. Jamilaldin Fatemi et al
potentially may incorporate manganese into
the porphyrin ring in place of iron, therefore
producing a manganese protoporphyrin instead of
haem [6]. Mn is known to be transported by the
transferrin receptor (TfR) and/or divalent metal
transporter (DMT1) [7]. Both transporters have
and plasma manganese are the readily available
biomarkers of manganese status in humans. The
strength of the observed correlation between
blood manganese concentrations and manganese
exposure concentrations often depends on the
magnitude and duration of exposure. In some
studies, whole blood manganese concentrations
correlate positively with exposure to manganese
[8]. In other cases, blood manganese concentrations
in exposed workers remain in the normal adult
-1), and urine and hair manganese
concentrations do not differ between exposed
and non-exposed workers [9]. In severe cases
of Mn poisoning, chelation therapy has been
recommended in order to reduce the body burden
of Mn. Chelation therapy that could be carried
out as a single and/or combined therapy involves
the use of chelating drugs that bind metal for the
treatment of potentially fatal conditions [10,11].
In this investigation, we studied the effect of MnCl2
exposure in low and high doses on blood serum
and hematology parameters in male Wistar rats.
Deferasirox (4-[3,5-bis (2- hydroxyphenyl)-1,2,4-
triazol-1-yl]-benzoic acid, or ICL670) is a tridentate
[12].
Deferiprone (1,2-dimethyl-3-hydroxypyrid-4-
one) is a bidentate chelator that was used in the
N
{5-[acetyl(hydroxy) amino]pentyl}-N-[5-({4-[(5-
aminopentyl)(hydroxy)amino]-4-oxobutanoyl}-
amino)pentyl]-N-hydroxysuccinamide) is a
hexadentate chelator. Manganese (Mn) used in
human body. Manganese ions enter to human body
from waters / foods (rice, apple, wheat, beans, lettuce)
and cause the different diseases. [13]. The high
values of manganese exist in spinach and tea leaves
in ranges of 10-100 mg kg-1 [14]. The manganese
at pH
7 and the oxidized forms archived in low pH [15,16].
Normal values of manganese in blood samples have
-1 [17]. The extra dosage of
manganese has toxicity effect and caused to many
diseases such as, memory impairment, dysfunction
on liver, renal and CNS. So, manganese in various
source such as water and blood samples must be
measured by analytical procedures. As toxicity
effect of manganese, it is caused to increase the
neutrophil count cells and decrease the red blood
cells. So, the various analytical techniques such
[18],
inductively coupled plasma mass spectrometry [19],
the inductively coupled plasma mass spectrometry-
MS [20] was used for determining of manganese
in biological samples. Also, due to the trace
the sample treatment used before sample analysis.
Many sample treatments such as, the liquid-liquid
extraction [21], the deep eutectic solvent extraction
[22], the column graphene oxide-based solid phase
extraction [23] were used for manganese extraction
in blood samples.
In this research, manganese ions were extracted
in blood of rats based on DFO, DFX and DFP
chelators by IL-LEM procedure and the manganese
concentration in rat blood samples was determined
by the F-AAS. Also, the toxic effects of manganese
on blood serum and hematology parameters such as:
RBC, WBC, HGB, PLT and HCT were investigated.
2. Experimental
2.1. Rats preparation
The Ethics Committee at Medical Sciences, The
University of Kerman, approved the research
protocol. Male Wistar rats were purchased from
Kerman Neuroscience Research Center, Kerman,
Iran. At the time of use the rats were 7–8 weeks
old, weighing 245±4g (mean± SEM.). All animals
were housed in well-cleaned sterilized cages
maintained in a room under controlled conditions
light; 12-h darkness; darkness: 7 p.m. – 7 a.m.) and
had free access to a standard diet and water.
80 Anal. Methods Environ. Chem. J. 4 (4) (2021) 78-91
2.2. Reagents
The desferrioxamine (DFO, CAS N.: 138-14-
7), deferasirox (DFX, CAS N.: 201530-41-8)
and deferiprone (DFP, CAS N.: 30652-11-0)
as chelating agents (ligands) were purchased
from Sigma Aldrich, Germany. 1-Hexyl-3-
methylimidazolium hexafluorophosphate (IL,
97.0% HPLC grade; CAS N.: 304680-35-1) was
prepared from Sigma Aldrich, Germany. Other
materials were prepared from Merck Chemical
Co. (Darmstadt, Germany). The standard solution
for manganese were prepared by dissolving of
1.0 g of Mn(NO3)2 in deionized water (DW, 1 Li).
The working standard solutions for calibration
of manganese were daily prepared by diluting
of standard solutions (1000 mg L-1) with DW
(Millipore, USA). All the glass and tubes were
cleaned by %10 nitric acid (v/v) for two days
and then washed by DW. The standard reference
materials (1640a; SRM) for manganese in water
) was used.
2.3. Instrumental
Flame atomic absorption spectrometer with a
double beam accessory (F-AAS, GBC 906, Aus.)
based on air-acetylene (C2H2), the D2 and HCL
lampas was used. The limits of detection (LOD)
and linear range was achieved 0.16 mg L-1 and
0.5-3.6 mg L-1, respectively by the FAAS. The
HCL light of manganese was optimized by two
screws (Hor. S and Ver. S) and the wavelength of
279.5 nm (slit of 0.2 nm; 5 mA) was selected for
F-AAS. After sample treatment, 1 mL of water
and blood samples were injected to burner of
FAAS by the auto-sampler. The working range
for F-AAS was obtained 0.2-3.8 mg L. The
validation of results was obtained by the graphite
furnace atomic absorption spectrophotometer
(GF-AAS, GBC, Aus.) for manganese analysis
in blood samples. The pH of the samples was
measured by Omega pH meter (USA). The
shaker (Grant, U.K) and centrifuge (speed 3000-
5000 rpm x g, Germany) was used for extraction/
separation of manganese from blood samples.
2.4. Chelation therapy design
In this study, manganese chloride [MnCl2 (LD50:
1715 mg kg-1)] at two doses of 30 mg kg-1 body
weight (low dose) and 60 mg kg-1 body weight
(high dose) was given to the drinking groups for
3 months followed by an early administration of
chelating agent. Study population consisted of 95
We assigned them randomly to control (5 rats) and
treated groups [high dose (45 rats) and low dose (45
rats)]. After 90 days, chelation therapy was carried
out after Mn application. Treated animals were
(Table 1): before chelation
therapy (5 rats), without chelation therapy [vehicle
oral (5 rats) and intraperitoneally (5 rats)], single
therapy (15 rats) and combined therapy (15 rats)
for both low and high doses. Chelators were given
orally (DFX and DFP) and intraperitoneally (DFO)
as single and combined therapies. Doses of DFX,
Table 1.
Treated groups (90):
Before chelation therapy
Without chelation therapy (Vehicle) Vehicle oral
Vehicle intraperitonealy
Single therapy DFX (30 mg kg-1 body weight)
DFO (30 mg kg-1 body weight)
L1 (60 mg kg-1 body weight)
Combined therapy DFX (15 mg kg-1 body weight)+DFO
(15 mg kg-1 body weigh
DFX (15 mg kg-1 body weight)+L1
(30 mg kg-1 body weight)
DFO (15 mg kg-1 body weight)+L1
(30 mg kg-1 body weight)
81
Manganese separation with ILLEM and toxicity evaluation S. Jamilaldin Fatemi et al
DFP and DFO were 30, 60 and 30 mg kg-1 body
weight, respectively (Table 1). Chelators were
given immediately after Mn application during 2
by exsanguinations from abdominal aorta and their
blood was collected for analysis.
2.5. Sample collection and analysis procedure
The full blood count sample was collected into a
citrate tube (Tek Lab, Catalogue No. K-100LS).
Hematology parameters such as RBC (106/l), WBC
(103 L), HGB (g dL), PLT (103 L) and HCT
(%) were measured in whole blood by countering
(Sysmex KX-21N) in hematology lab.
By the IL-LEM procedure, the DFO, DFX and DFP
chelators mixed with IL and used for speciation and
extraction of Mn ions in rat blood samples (Fig.1).
The chelators dispersed in 10 mL of standard
-1) and rat blood
samples, after chelation and extraction Mn ions, the
hydrophobic ionic liquid ([HMIM] [PF6];100 mg)
was added to samples and diluted with acetone. In
fact, the Mn ions was extracted by coordination
bond of nitrogen at optimized pH (98%), but at
higher pH, manganese ions precipitated (Mn(OH)2).
The manganese chelated with the DFO, DFX and
DFP chelators and separated from samples by
hydrophobic ionic liquid ([HMIM] [PF6]) in end
of conical tube after centrifuging for 5 min at 3500
rpm. After removing upper blood/serum phase,
manganese ions were back-extracted from the
DFO/IL, DFX/IL and DFP/IL into aqueous phase
with 0.3 mL of HNO3 solutions (0.2M). Finally, the
resulting solution was determined by F-AAS after
dilution with DW up to 0.5 mL. The linear range
-1 or 0.025-0.18
mg L-1 by the IL-LEM procedure.
2.6. Statistical analysis
The statistical package for the sciences (SPSS,
Bristol, England) version 18 was used to process
the data. All comparisons among the groups were
analyzed with a one-way analysis of variance
(ANOVA) followed by LSD test for multiple
comparisons. Data were reported as the mean±SEM.
In all comparisons, p<0.05 was the criterion for
Fig.1. Determination of manganese in the rat blood/serum based on chelators
by the IL-LEM procedure coupled to F-AAS
82 Anal. Methods Environ. Chem. J. 4 (4) (2021) 78-91
3. Results and Discussion
3.1. Symptoms
Over 90 days, results indicated that difference weight
both the high and low doses groups compared to
the control group (Table 2). Furthermore, toxicity
symptoms such as: skin reaction, black spots on
liver and bright lung appeared.
3.2. Hematology parameters
The hematology parameters are shown in Table 3
and 4. Compared to each respective control group, a
both low and high doses groups. The RBC, WBC,
high dose group (Table 3 and 4, Fig. 2). In order to
investigate the effect of passing time in removing
Mn from the body spontaneously, one group was
treated as without chelation therapy (vehicle).
between the vehicle and before chelation therapy
groups, results of chelation therapy were compared
to each respective vehicle group. After chelation
DFO+DFX in low dose group and DFO in high
by DFX, DFP(L1) and DFO+DFX in low dose
group and DFO, DFP (L1), DFX, DFO+DFP
(L1), DFO+DFX and DFX+DFP(L1) in high dose
group. The PLT count by DFO and DFX+DFP(L1),
also HCT count by DFO and DFO+DFP (L1)
(Table 3
and 4, Fig. 3)
was set as p<0.05)
Table 2. Bodyweights over 90 days for rats in different groups (all data are expressed as the mean±SEM)
High dose
drinking of manganese
Low dose
drinking of manganese
ControlGroup
244.61 ± 4.20
286.61 ± 7.95
249.80 ± 2.91
299.60 ± 4.55
242.21 ± 8.62
297.20 ± 7.46
Initial body weight (g)
Final body Weight (g)
Table 3. Hematology parameters in low-dose groups (all data are expressed as the mean±SEM)
Chelation
therapy
DFX+DFP
(L1)
Chelation
therapy
DFX+DFO
Chelation
therapy
DFO+DFP
(L1)
Chelation
therapy
DFO
Chelation
therapy
DFP
(L1)
Chelation
therapy
DFX
Vehicle
Before
chelation
therapy
Control Group
7.63±0.16 6.98±0.57 7.88±0.147.75±0.117.28±0.427.58±0.337.91±0.39 7.28±0.11 7.24±0.25
RBC
(106 L-1)
8.04±0.83 12.25±1.22 9.80±1.64 9.80±0.84 11.10±1.14 9.94±0.59 9.79±1.948.60±1.947.42±0.43
WBC
(103 L-1)
12.18±0.33 10.80±2.1812.15±0.43 12.77±0.14 11.35±0.03 11.54±0.89 12.75±0.38 13.02±0.75 11.96±0.29
HGB
(g dL-1)
658.80±71.39 579.40±153.65691.40±48.52654.20±69.69828.00±60.44782.00±37.83 663.20±45.28680.02±47.69 501.79±45.53
PLT
(103 L-1)
39.42±0.59 35.68±2.7338.96±0.6741.00±0.2637.62±1.7237.70±1.5538.62±1.1637.39±0.2039.02±1.21 HCT (%)
1.19±0.071.34±0.171.02±0.201.01±0.151.08±0.121.32±0.141.79±0.221.95±0.151.28±0.10
Mn
concentration
(mg L-1)
ANOVA analysis shows p value in all of treatment groups in comparison to vehicl
83
Manganese separation with ILLEM and toxicity evaluation S. Jamilaldin Fatemi et al
Table 4. Hematology parameters in high-dose groups (all data are expressed as the mean±SEM).
Chelation
therapy
DFX+L1
Chelation
therapy
DFX+DFO
Chelation
therapy
DFO+
(DFP) L1
Chelation
therapy
DFO
Chelation
therapy
L1(DFP)
Chelation
therapy
DFX
Vehicle
Before
chelation
therapy
ControlGroup
8.41±0.18 7.74±0.18 7.87±0.307.30±0.217.92±0.708.08±0.158.30±0.83 8.04±0.12 7.24±0.25
RBC
(106 L-1)
8.74±1.41 11.32±1.08 9.42±1.02 11.56±1.38 15.28±2.40 10.76±1.40 10.74±1.1311.22±1.447.42±0.43
WBC
(103 L-1)
12.84±0.29 12.17±0.3212.38±0.29 11.50±0.46 12.68±0.20 12.34±0.39 13.80±0.22 14.27±0.22 11.96±0.29
HGB
(g dL-1)
594.25±140.02723.66±42.03806.00±67.66623.00±68.40800.40±46.70630.25±56.54 806.75±42.74 824.00±48.12 501.79±145.53
PLT
(103 L-1)
42.60±0.96 39.70±0.6338.95±0.6738.08±1.1040.86±0.5942.85±0.7040.95±0.8840.30±0.5839.02±1.21
HCT
(%)
1.18±0.091.28±0.211.23±0.141.14±0.161.42±0.051.32±0.141.32±0.142.03±0.261.28±0.10
Mn
concentration
(mg L-1)
ANOVA analysis shows p value in all of treatment groups in comparison to vehicl
Manganese separation with ILLEM and toxicity evaluation
*Corresponding Author: S. Jamilaldin Fatemi
Email: fatemijam@uk.ac.ir
https://doi.org/10.24200/amecj.v4.i04.160
Fig. 2. Hematology parameters and Mn concentrations in control, high and low doses of Mn
administration (Before chelation therapy) , *p<0.05, **p<0.01, ***p<0.001 as compared to control.
0
2
4
6
8
10
12
14
Control L.D H.D
WBC(103/Lit)
*
0
100
200
300
400
500
600
700
800
900
1000
Control L.D H.D
PLT (103/Lit)
*
0
0/5
1
1/5
2
2/5
Control L.D H.D
Mn concentration (mg/Lit)
** *
6/4
6/6
6/8
7
7/2
7/4
7/6
7/8
8
8/2
8/4
Control L.D H.D
RBC(106/Lit)
**
0
2
4
6
8
10
12
14
16
Control L.D H.D
HGB
*
***
35
36
37
38
39
40
41
42
Control L.D H.D
HCT(%)
*
Manganese separation with ILLEM and toxicity evaluation
*Corresponding Author: S. Jamilaldin Fatemi
Email: fatemijam@uk.ac.ir
https://doi.org/10.24200/amecj.v4.i04.160
Fig. 2. Hematology parameters and Mn concentrations in control, high and low doses of Mn
administration (Before chelation therapy) , *p<0.05, **p<0.01, ***p<0.001 as compared to control.
0
2
4
6
8
10
12
14
Control L.D H.D
WBC(103/Lit)
*
0
100
200
300
400
500
600
700
800
900
1000
Control L.D H.D
PLT (103/Lit)
*
0
0/5
1
1/5
2
2/5
Control L.D H.D
Mn concentration (mg/Lit)
** *
6/4
6/6
6/8
7
7/2
7/4
7/6
7/8
8
8/2
8/4
Control L.D H.D
RBC(106/Lit)
**
0
2
4
6
8
10
12
14
16
Control L.D H.D
HGB
*
***
35
36
37
38
39
40
41
42
Control L.D H.D
HCT(%)
*
Manganese separation with ILLEM and toxicity evaluation
*Corresponding Author: S. Jamilaldin Fatemi
Email: fatemijam@uk.ac.ir
https://doi.org/10.24200/amecj.v4.i04.160
Fig. 2. Hematology parameters and Mn concentrations in control, high and low doses of Mn
administration (Before chelation therapy) , *p<0.05, **p<0.01, ***p<0.001 as compared to control.
0
2
4
6
8
10
12
14
Control L.D H.D
WBC(103/Lit)
*
0
100
200
300
400
500
600
700
800
900
1000
Control L.D H.D
PLT (103/Lit)
*
0
0/5
1
1/5
2
2/5
Control L.D H.D
Mn concentration (mg/Lit)
** *
6/4
6/6
6/8
7
7/2
7/4
7/6
7/8
8
8/2
8/4
Control L.D H.D
RBC(106/Lit)
**
0
2
4
6
8
10
12
14
16
Control L.D H.D
HGB
*
***
35
36
37
38
39
40
41
42
Control L.D H.D
HCT(%)
*
Fig. 2. Hematology parameters and Mn concentrations in control, high and low doses
of Mn administration (Before chelation therapy) , *p<0.05, **p<0.01, ***p<0.001 as compared to control.
84 Anal. Methods Environ. Chem. J. 4 (4) (2021) 78-91
Manganese separation with ILLEM and toxicity evaluation
*Corresponding Author: S. Jamilaldin Fatemi
Email: fatemijam@uk.ac.ir
https://doi.org/10.24200/amecj.v4.i04.160
Fig. 3. Hematology parameters and concentrations of Mn in Vehicle and treated groups after
chelation therapy, *p<0.05, **p<0.01, ***p<0.001 significantly different from HD vehicle and
^p<0.05, ^^p<0.01, ^^^p<0.001 significantly different from LD vehicle, by one-way ANOVA
followed by LSD’s multiple comparison test.
0
1
2
3
4
5
6
7
8
9
10
Vehicle L1+DFX DFO+DFX DFO+LI DFO L1 DFX
RBC(106/Lit
HD LD
^
*
0
2
4
6
8
10
12
14
16
18
20
Vehicle L1+DFX DFO+DFX DFO+LI DFO L1 DFX
WBC(103/Lit
HD LD
^^ *
0
100
200
300
400
500
600
700
800
900
1000
PLT (103/Lit)
HD LD
**
0
2
4
6
8
10
12
14
16
Vehicle L1+DFX DFO+DFX DFO+L1 DFO L1 DFX
HGB
HD LD
*
*
*
*
**
*
^
^
^
0
0/5
1
1/5
2
2/5
Mn concentration (mg/lit)
HD LD
*^
**
^
**
^^
**
^
*
^^
**
^
0
5
10
15
20
25
30
35
40
45
50
HCT (%)
HD LD
*
**
Fig. 3. Hematology parameters and concentrations of Mn in Vehicle and treated
vehicle, by one-way ANOVA followed by LSD’s multiple comparison test
85
Manganese separation with ILLEM and toxicity evaluation S. Jamilaldin Fatemi et al
3.3. Mn concentration
Administration of manganese chloride in both of
increase in concentration of Mn (Table 3 and 4,
Fig. 2). After chelation therapy, all chelatores
and high doses groups. Although DFO was more
effective chelator (Table 3 and 4, Fig. 3)
3.4. Optimization of extraction parameters
samples, the effect of parameters such as the amount
of ligand, the pH, the Ionic liquids, the shaking
and centrifuging time, the sample volume and the
interferences ions were studied and optimized.
3.4.1.pH effect
The effect of pH on extraction of manganese in
water and rat blood samples must be optimized.
The pH effect on the manganese complexation
by the DFO, DFX and DFP chelators. So, the
various pH between 2 - 11 was evaluated for the
Mn extraction in rat blood samples. The pH was
controlled by a buffer solution. The result showed,
the high recovery based on chelators for manganese
was obtained at pH
of 7. The recoveries were decreased for Mn at pH
ranges less than 7 and more than 8.0. So, the pH of
7.0-7.5 were selected for extraction of manganese
in waters and rat blood samples by the IL-LEM
procedure (Fig. 4).
3.4.2. Optimization of DFO, DFX and DFP
chelators
The concentration of chelators is main parameters
for manganese extraction which must be optimized
by the IL-LE procedure. For optimizing, 0.1×10–
0.9×10 mol L of DFO was used in the rat blood
sample. The results showed that, by increasing
ligand concentration up to 0.6×10 mol L, the
recoveries are also increased (Fig. 5). So, the
amount of chelating agent (DFO) between 0.6-0.9
-1-1 was
found the best amount for Mn extraction. The DFX
and DFP have almost used with the similar range
-1.
Fig.4. The effect of pH on manganese extraction by IL-LE method
Manganese separation with ILLEM and toxicity evaluation
*Corresponding Author: S. Jamilaldin Fatemi
Email: fatemijam@uk.ac.ir
https://doi.org/10.24200/amecj.v4.i04.160
Fig.4. The effect of pH on manganese extraction by IL-LE method
3.4.2. Optimization of DFO, DFX and DFP chelators
The concentration of chelators is main parameters for manganese extraction which must be
optimized by the IL-LE procedure. For optimizing, 0.02 × 10−5 – 0.5 × 10−4 mol L−1 of DFO was
used in the rat blood sample. The results showed that, by increasing ligand concentration up to
0.07 × 10−5 mol L−1, the recoveries are also increased (Fig. 5). So, the amount of chelating agent
(DFO) between 0.7-0.9 μmol L-1 was investigated and 0.8 μmol L-1 was found the best amount for
Mn extraction. The DFX and DFP have almost used with the similar range between 0.6-1 μmol L-1.
0
20
40
60
80
100
120
۲۳۴۵۶۷۷/۵ ۸ ۹ ۱۰
Recovery (%)
pH
Rat blood Water
2 3 4 5 6 7 7/5 8 9 10
86 Anal. Methods Environ. Chem. J. 4 (4) (2021) 78-91
3.4.3. Optimization of sample volume and eluent
The sample volume of rat blood samples and the
standard solution was evaluated from 1.0 mL to
25 mL for Mn concentration between 25-180 µg
L-1
was obtained for 10 mL of rat blood samples at pH
7-7.5 (Fig. 6). In-addition, the effect of eluent on
manganese extraction based on ligand/IL (DFO,
DFX and DFP/IL) were evaluated. At low pH, the
covalent bond between the manganese and nitrogen
group was break-down and released the Mn ions
into acid phase. So, the acid solutions (HCl, HNO3,
H2SO4) were used for back-extraction process in
blood and water samples. The results showed, the
3 (0.2 M,
0.3 mL) (Fig. 7).
Fig. 5. The effect of ligand on manganese extraction by IL-LE method
Fig. 6. The effect of sample volume on manganese extraction by IL-LE method
Manganese separation with ILLEM and toxicity evaluation
*Corresponding Author: S. Jamilaldin Fatemi
Email: fatemijam@uk.ac.ir
https://doi.org/10.24200/amecj.v4.i04.160
Fig. 6. The effect of sample volume on manganese extraction by IL-LE method
Fig. 7. The effect of eluents on manganese extraction by IL-LE method
0
20
40
60
80
100
120
۲۴۶۸۱۰ ۱۲ ۱۵ ۲۰ ۲۵
Recovery (%)
Sample volume (mL)
Rat blood water
0
20
40
60
80
100
120
۰/۰۵ ۰/۱ ۰/۱۵ ۰/۲ ۰/۲۵ ۰/۳ ۰/۵
Recovery (%)
Eluent (mol)
HCl HNO3 H2SO4
2 4 6 8 10 12 15 20 25
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
87
Manganese separation with ILLEM and toxicity evaluation S. Jamilaldin Fatemi et al
3.5. Discussion of Manganese toxicity
By procedure, the manganese extracted based
on ligand and IL in rat blood samples before
determined by the F-AAS. Also, the toxicity of
manganese in rat evaluated by chelation therapy
with DFO, DFX and DFP before determined
by the IL-LE procedure at pH=7-7.5. Due to
biologically evaluation, manganese is considered
to be an essential metal important to mitochondrial
oxidative processes for all living mammals, but
may also be toxic at high concentrations [24].
Manganese toxicity seems to be largely due to Mn
induced cellular free radical damage [25]. This
hypothesis is based on the potent redox properties
of Mn. In our current study uptake of manganese
after oral exposure led to elevating RBC, WBC,
HGB, PLT and HCT. Furthermore, we observed
a clear increase in level of Mn in blood serum
as compared to control. According the previous
study, Mn in the blood compartment may tend
to accumulate in the blood cells [26]. RBC count
blood manganese [6]. Hemoglobin is a protein
containing iron in red blood cells. One red blood
cell contains 280 million molecules of hemoglobin.
Exposure to Mn during the toxicity period, results
increase in both counts of RBC and HGB in whole
blood. Manganese leaving the enterocyte and
entering the circulation bound to transferrin, may
bind to transferrin receptors on erythroid cells.
The erythroid cell potentially may incorporate
manganese into the porphyrin ring in place of iron,
therefore producing a manganese proto-porphyrin
instead of hemoglobin [6]. Subsequently, the
amount of deformed hemoglobin increases.
Hematocrite is the percentage of red blood cells.
Therefore, after toxicity with Mn, its behavior is
similar red blood cells. The platelet is responsible
for blood coagulation. Interestingly, Mn toxicity
increase platelet and the immune system become
more active by increasing white blood cell counts.
A simple measurement of extracellular Mn such
Mn concentrations in the blood compartment,
including blood cells, so whole blood must be
analyzed [26]. However, Serum levels of metal
are questioned as valid markers of total-body
of metal. In any event, our results indicate that
Mn can accumulate in serum. In severe case
of manganese poisoning, chelation therapy has
been recommended in order to reduce the body
burden of manganese. Deferasirox, deferipron
and desferrioxamine are commonly used chelating
agent for treatment of a variety of metal-induced
Fig. 7. The effect of eluents on manganese extraction by IL-LE method
Manganese separation with ILLEM and toxicity evaluation
*Corresponding Author: S. Jamilaldin Fatemi
Email: fatemijam@uk.ac.ir
https://doi.org/10.24200/amecj.v4.i04.160
Fig. 6. The effect of sample volume on manganese extraction by IL-LE method
Fig. 7. The effect of eluents on manganese extraction by IL-LE method
0
20
40
60
80
100
120
۲۴۶۸۱۰ ۱۲ ۱۵ ۲۰ ۲۵
Recovery (%)
Sample volume (mL)
Rat blood water
0
20
40
60
80
100
120
۰/۰۵ ۰/۱ ۰/۱۵ ۰/۲ ۰/۲۵ ۰/۳ ۰/۵
Recovery (%)
Eluent (mol)
HCl HNO3 H2SO4
0.50.30.250.20.150.10.05
88 Anal. Methods Environ. Chem. J. 4 (4) (2021) 78-91
toxicities [27, 28], especially treatment of
iron. In our previous studies, it has been shown
these chelators increase toxin elimination and
reducing the body burden of manganese [29,
30]. Also current our study supports the clinical
effectiveness of these chelators. Results show
that the above chelators are able to return Mn and
hematology parameters to nearly normal level
of control group. On the other hand, they can
determine/separate/extraction of Mn ions in rat
blood samples with high accuracy and precision
by F-AAS. The results showed, the DFO as single
and combined with DFX and L1 is more effective
for extraction of manganese ions. Surprisingly
after chelation therapy by combination of DFX
and DFO count of WBC increased. There is no
satisfactory explanation for this result. However, it
is reasonable to postulate that the immune system
against the drug increases the white blood cell
count.
3.6. Validation of extraction procedure
By the ILLE procedure, the separation and
determination of Mn ions in rat blood samples
were achieved at pH from 7.0 to 7.5. In the real
samples such as, water, rat blood and rat serum, the
result of manganese were validated by spiking to
standard solutions of manganese (10, 20, 40. 60 µg
L-1) at optimized conditions (Table 5). The results
was carried out in real samples based on mixture of
DFX, DFO, L1 and [HMIM][PF6] before shaking
and centrifuging process. Finally after extraction
and back-extraction of Mn ions, the remained
solution was determined by F-AAS by dilution of
DW up to 0.5 mL.
4. Conclusions
In summary, the speciation and determination of
Mn ions in rat blood samples were achieved by the
ILLE procedure at pH from 7.0 to 7.5. The three
chelators, DFX, DFO and L1 used as chelation
therapy in rats, then the [HMIM][PF6] added to
blood of rats. After shaking, extraction, centrifuging
and back-extraction, the Mn values were determined
by F-AAS. The linear range and LOD was obtained
-1 -1, respectively. Also,
Table 5. Validation of ILLEM for determination of manganese ions in water, whole blood, serum
; n=8)
Samples Added *Found Mn *Final concentration Mn
Dilution factor 20 Recovery (%)
Whole blood of rat ----- 6.54 ± 1.11 530.80 ± 25.51 -----
20 45.83 ± 2.05 916.60 ± 40.42 96.5
40 68.12 ± 2.88 1362.40 ± 53.72 103.9
Serum of rat ----- 13.68 ± 0.62 273.60 ± 12.76 -----
10 23.14 ± 1.12 462.80 ± 21.73 94.6
20 33.52 ± 1.69 670.40 ± 29.82 99.2
Plasma rat ----- 7.68 ± 0.33 153.60 ± 6.54 -----
5 12.55 ± 0.56 251.00 ± 11.75 97.4
10 17.97 ± 0.78 359.40 ± 16.43 102.9
Water ----- 23.88 ± 1.12 ----- -----
20 43.64 ± 1.98 ----- 98.8
40 62.41 ± 2.95 ----- 96.3
*x ± ts /√n at 95% condence (n=8)
1 mL of Whole blood, Serum and plasma diluted with DW up to 20(1:20)
89
Manganese separation with ILLEM and toxicity evaluation S. Jamilaldin Fatemi et al
the results of the current study clearly demonstrated
that chronic Mn exposure not only resulted in a
marked increase of Mn concentrations in blood
of RBC, WBC, HGB, PLT and HCT in whole
blood. Chelation therapy was effective in returning
hematology parameters and Mn ions to nearly
normal level. As well as three chelators are more
in removing manganese from tissues. Therefore,
after basic preclinical research this could be
recommended for human administration.
5. Acknowledgements
The authors wish to thanks from the head and
director of Kerman Neuroscience Research
Center, Mohammad Faghihi Zarandi as statistical
data analyzer, and Shahid Bahonar University
of Kerman Faculty Research for Funding and
supporting of this research.
6. Declaration of interest
authors alone are responsible for the content and
writing of this article.
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