Spectrochimica Acta Part B 61 (2006) 1219 1223

www.elsevier.com/locate/sab

Analytical note

Cognitive impairment related changes in the elemental concentration in the

brain of old rat
R.F.B. Serpa a,, E.F.O. de Jesus b , M.J. Anjos a,b , R.T. Lopes a , M.G.T. do Carmo c , M.S. Rocha d ,
L.C. Rodrigues d , S. Moreira e , A.M.B. Martinez f
a

Federal University of Rio de Janeiro/COPPE, Nuclear Instrumentation Laboratory, P.O. Box: 68509, Zip Code: 21941-972, Rio de Janeiro, Brazil
b
University of Rio de Janeiro State, Physics Institute, RJ, Brazil
c
Federal University of Rio de Janeiro, Nutrition Institute, RJ, Brazil
d
Federal University of Rio de Janeiro, Department of Basics and Clinic Pharmacy, RJ, Brazil
e
University of Campinas State, Civil Engineering Department, SP, Brazil
f
Federal University of Rio de Janeiro, Department of Histology and Embryology, RJ, Brazil
Received 13 December 2005; accepted 22 August 2006
Available online 11 October 2006

Abstract
In order to evaluate the elemental concentration as a function of learning and memory deficiency, six different structures of the brain were
analyzed by total reflection X-ray fluorescence spectrometry with synchrotron radiation (SR-TXRF). To evaluate the cognitive processes, the
animals were tested in an adaptation of the Morris water maze. After the test, the animals were divided into two groups: cognitively healthy
(control group) and cognitively impaired. The measurements were carried out at XRF beam line at Light Synchrotron Brazilian laboratory,
Campinas, Brazil. The following elements were identified: Al, P, S, Cl, K, Ca, Ti, Cr, Fe, Cu, Zn, Br and Rb. K concentration was higher in all
regions of the brain studied for control group than the cognitively impaired group. Moreover, the control group presented higher levels for P and
Fe in the entorhinal cortex, in the temporal cortex (only P), in the hypothalamus and in the thalamus, than the cognitively impaired group. Br
concentration in the animals which presented cognitive impairment was three times larger in the hypothalamus and thalamus, twice larger in
temporal cortex and higher in visual cortex than the cognitively healthy group. Cu was more remarkable in the hippocampus and hypothalamus
from the animals with cognitive impairment than the control group.
We observed that the cognitively impaired group presented highest concentrations of Br and Cu in certain areas than the control group, on the
other hand, this group presented highest levels of K for all brain areas studied.
2006 Elsevier B.V. All rights reserved.
Keywords: Brain; Trace element; Learning; Memory; SR-TXRF

1. Introduction
It is known that aging is associated with neurobehavioral
deficits [1]. The aging process of the human brain is
characterized by progressive neuronal loss [2]. Certain brain
areas are more vulnerable to neuronal degeneration than others,

This paper was presented at the 11th International Conference on Total

Reflection X-ray Fluorescence Spectrometry and Related Methods (TXRF2005), held in Budapest, Hungary, 1822 September 2005, and is published in
the special issue of Spectrochimica Acta Part B, dedicated to that conference.
Corresponding author. Tel.: +55 21 25627311.
E-mail address: renata@lin.ufrj.br (R.F.B. Serpa).
0584-8547/$ - see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.sab.2006.08.005

reflecting an altered resistance to stress of the tissue itself and/or

the lack of adequate immunological defense mechanisms in
these regions. Calcium and iron are mediators of the aging
process in the normal brain. Enhanced Ca levels are related to
apoptosis a process possibly involved in the brain aging [3].
Moreover, literature reports significant changes in mineral
status with age [4] as differences in the interaction between
toxic metals and some essential elements [1]. It has been shown
that excess concentrations of a number of elements in the brain
are capable of producing harmful effects by displacing some
essential elements, while in turn, numerous toxic and essential
elements have been reported to be imbalanced in Alzheimer
Disease (AD) [1].

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R.F.B. Serpa et al. / Spectrochimica Acta Part B 61 (2006) 12191223

Fig. 1. A view of the aquatic labyrinth.

One of the critical aspects of aging, of any living organism, is

the decrease in its ability to adapt to changes in its environment,
and as the cognitive impairment is one of the more severe
outcomes of human aging [5], this study may have an important
contribution to understand how the mineral concentrations are
related to cognitive performance.
Therefore, in order to evaluate the elemental concentration as
a function of learning and memory deficiency, six different
structures of the brain were analyzed by the total reflection Xray fluorescence spectrometry with synchrotron radiation (SRTXRF).

within 120 s, the animal was placed on the platform for 10 s and
then removed from the pool. The rat was given five trials per
day during 4 days with an inter-trial interval of 40 min (Fig. 2).
The location of the escape platform remained unchanged, but
the point of entry of the rat into the pool was different on each
trial. After 10 days, animals were retested in order to evaluate
their long-term memory (retention test). An animal was
considered cognitively healthy when it takes approximately
20 s to find the platform.

2. Experimental

The animals were sacrificed by decapitating and their brains

were quickly and carefully removed and immediately frozen in
liquid nitrogen. Subsequently, six structures were dissected for
trace and major elements analysis, i.e., the temporal cortex,
entorhinal cortex, visual cortex, hippocampus, thalamus and
hypothalamus, 50 mg of each sample, were submitted to
chemical digestion by adding 0.5 mL of nitric acid (HNO3
65%, analytical grade from Merck) for 4 h at 60 C. After the
dissolution, the samples were mixed with 50 L of ICP standard
gallium solution, which was used as an internal standard. Small
amount (8 L) of solution was pipetted out on a clean Perspex
sample support. Each sample was dried in infrared spot. All
samples were processed in triplicates. Moreover, blank samples
were done in order to evaluate any source of contamination.

2.1. Animals
Twelve female Wistar rats, 24 months old, obtained from the
animal facility of the Nutrition Institute of Federal University of
Rio de Janeiro, were used in this study following approved
guidelines for animal care. Animals were allowed to acclimatize to the behavioral experimental room for at least 4 days
before procedures began, in a temperature-controlled environment (20 2 C), relative humidity, with lights on from 6 a.m.
to 6 p.m. (12 h-diurnal light/12 h-dark cycles) and ad libitum
access to commercial pellet food and tap water, 4 rats per cage.
All animals were submitted to an adaptation of the Morris
water maze, in order to evaluate their cognitive performance.

2.3. Sample and standard preparations

2.2. Spatial memory test

Spatial memory in rats was assessed using an adaptation of
the Morris water maze [6] carried out in the same behavioral
room in which the rats were acclimatized under the conditions
of temperature, humidity and light cycle described above. A
circular pool (Fig. 1) (180 cm in diameter, 50 cm high) was
filled to a depth of 30 cm with opaque water at a temperature of
20 2 C, and divided into four quadrants of equal area. A
platform with 8 cm diameter was placed 1 cm below the water
surface, midway between the center and the rim of the pool in
one of the quadrants. The rat was placed into the pool facing the
rim, and its escape latency, time taken by the rat to find the
platform, measured by an observer. If the rat failed to find it

Fig. 2. Diagram of the different position that the animals are liberated in the
swimming pool.

R.F.B. Serpa et al. / Spectrochimica Acta Part B 61 (2006) 12191223

Fig. 4. Characteristic X-ray spectrum of a brain sample.

Fig. 3. Detection limits for the brain samples using SR-TXRF.

To set up the calibration curve, five standard solutions

containing the elements: Al, Si, Ca, Ti, Cr, Fe, Ni, Zn, As, Se,
Sr, Mo for K-lines and Mo, Ba, Sm, Lu, Pt, Tl and Pb for L-lines
were prepared at different and known concentrations, with Ga
addition as the internal standard.

1221

2.5. Statistical analysis

The results were expressed as mean values standard error.
The results from each type brain structures of the two groups
analyzed were compared using analysis of Student t-test with
5% significance level.

2.4. TXRF analysis

3. Results and discussion
Since the introduction of the TXRF spectrometry, there has
been a continuous improvement in excitation and detection methods leading a high degree of development. The advent of
synchrotron radiation (SR) sources became possible new advances
in the application of XRF to biological systems [7]. One of the
advantages of this technique is the small sample volume required
for the analysis few L for liquid samples and few g for solid
samples [8], therefore the sample can be considered as a thin film,
which facilitate the quantification analysis, since the effects of
absorption and enhancement can be neglected.
In the TXRF, the quantification is usually carried out by
internal standardization method. This method is based on the
addition of an element that is not present in the multielementar
sample, for example galium (Ga). It is useful because the thin
film formed on the Perspex support does not have regular
geometry and the X-ray intensities depend on the thin film
position. This geometry effect [9] can be corrected by
normalization of each element X-ray line in relation to an
internal standard added in every sample and standard.
The measurements were performed at the Light Synchrotron
Brazilian laboratory (LNLS), Campinas, So Paulo, Brazil. The
sample carrier was placed in a horizontal plane in relation to the
HPGe detector (140 eV at 5.9 keV) and excited by a white beam
of irradiation with a maximum energy of 20 keV filtered by
0.5 mm of aluminum, with an angle of incidence of 1.0 mrad.
The samples and the standards were excited for 100 s and, the
X-ray spectra obtained were evaluated by the software
Quantitative X-ray Analysis System (QXAS) [10], distributed
by the International Atomic Energy Agency (IAEA), in order to
obtain the X-ray intensities for each element and the associated
uncertainty.

The detection limit (DL) was determined according to

Ladisich et al. [11]. Fig. 3 shows the DL, which was calculated
using an extrapolated measuring time of 1000 s.
Fig. 4 shows X-ray characteristic spectra from brain samples
using TXRF.
The accuracy of the measurements was checked by
determining the elemental concentrations in a standard solution
prepared with bovine liver NIST1577b, a certified material
from National Institute of Standards and Technology (NIST), as
shown in Table 1.
The basal prefrontal area and the temporal lobe share
thalamic and cortical projections which are engaged in different
functions for recall. Most likely, the prefrontal cortex will be a
control center for metamemory, effortful initiation of recall, and
the temporary sequencing and organization of information [12].
It is well documented that the prefrontal cortex is the most
Table 1
Elemental concentration (g g 1) in a certified material Bovine Liver,
NIST1577b
Elements

Certificate concentration

Measurement concentration

P
S
Cl
K
Ca
Mn
Fe
Cu
Zn
Rb

11000 300
7850 60
2780 60
9940 20
116 4
10.5 1.7
184 15
160 8
127 16
13.7 1.1

9130 40
6235 25
580 10
8620 20
115 6
7.7 0.2
170 1
135 0
113 0
15 1

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R.F.B. Serpa et al. / Spectrochimica Acta Part B 61 (2006) 12191223

Table 2
Elemental concentration (g g 1) in entorhinal, temporal and visual cortex from the brain of old female Wistar rats
Elements

Entorhinal cortex
Normal group

Al
P
S
Cl
K
Ca
Fe
Cu
Zn
Br
Rb

2270 750
1580 140
1520 70
440 30
3530 20
270 4
50 1
4.8 0.2
35.2 0.3
bDL
51

Temporal cortex
C.I. group

Normal group
a

3180 695
1440 130
1450 80
510 40
3410 25
360 5
42.7 0.3
4.2 0.1
31.9 0.3
bDL
51

Visual cortex
C.I. group

Normal group
a

2490 625
1520 170
1110 50
440 20
3400 30a
305 3
43 0.3a
4.0 0.1
33.1 0.2
4 1a
51

4000 1000
1410 160
1100 80
470 40
2750 20a
290 4
75 1a
3.9 0.2
30.6 0.2
8 0.3a
41

3810 960
1460 180
1720 90
760 40
3250 25a
600 10a
95 1a
4.6 0.2
35.5 0.3
41
bDL

C.I. group
2600 680a
1640 150
1470 50
440 30
3000 20a
360 4a
80 1a
3.8 0.1
34.6 0.3
61
51

Values are means standard error. The subscript letter a indicate differences statistically significant between the groups, in each region at P 0.05.

amydalar, and perirhinal regions are prominent nodes in this

glutaminergic network, it is presumed that vesicular Zn2+ is
involved in modulation of neuronal excitability and in the synaptic
plasticity of developmental and experiential learning [15].
The Fe accumulation can result in oxidative damage and
neurodegeneration [16]. In the cognitively impaired animal
group an increase of iron was observed in the hippocampus,
which is a brain region that is directly involved in the learning
and memory.
Moreover, the Br concentration was more pronounced in all
brain structures of the cognitively impaired group. This can be
explained since studies reported an increase of Br in the cortex
and hippocampus in the Alzheimer disease [17].

sensitive to the negative effects of aging among the brain

regions, but it is unclear whether aging affects brain structure
uniformly across all prefrontal regions. In addition to structural
vulnerability, behaviors associated with prefrontal regions are
among the most sensitive to negative effects of aging. Both
cross-sectional and longitudinal studies suggest alterations in
such prefrontal functions as working memory, complex problem
solving, concept formation and inhibition of response [13].
The elemental concentration can be seen on Tables 2 and 3.
Discussing these tables, we can observe that there are statistically significant differences in the elemental concentration
between the impaired group and the normal one.
The animals that presented cognitive impairment showed
highest levels of Al in the cortical area enthorinal and
temporal cortex, hippocampus and hypothalamus. Aluminum is
a highly neurotoxic element and can result in degeneration of
nerve cells in the brain of humans and experimental animals. It
has been reported that Al is involved in the etiology of dialysis
dementia, amyotrophic lateral sclerosis and Alzheimer's disease
[14].
The hippocampus of cognitively impaired group presented
higher levels of Cl, Cu and Fe (temporal cortex) than the normal
one. They also presented higher concentrations of Zn in the
hypothalamus than the normal one. As the hippocampal,

4. Conclusions
The hippocampus is a brain area which is involved in the
memory and learning mechanisms. In this study we observed
that some elements, such as Al, Fe, Cu, Zn and Br presented
higher concentrations in samples from animals that presented
cognitive impairment. Some of these elements are important for
the normal function of organisms, however high levels are also
associated with degenerative disorders like Alzheimer's disease, Parkinson's disease and Huntington's disease.

Table 3
Elemental concentration (g g 1) in hippocampus, thalamus and hypothalamus from the brain of old female Wistar rats
Elements

Hippocampus
Normal group

Al
P
S
Cl
K
Ca
Fe
Cu
Zn
Br
Rb

1790 400
1550 110
1260 60
284 30
2740 20a
195 4
30.9 0.2a
4.4 0.1a
27.9 0.2
bDL
61

Thalamus
C.I. group

Hypothalamus

Normal group
a

2680 630
1510 130
1070 70
400 30
1760 20a
210 4
42.4 0.4a
6.3 0.2a
31.0 0.3
2.6 0.5
61

6850 1850
2650 260a
3050 140a
660 60
4500 30a
330 4
152 1
5.3 0.1
29.1 0.2
bDL
41

C.I. group

Normal group
a

11300 1860
1380 190a
1000 90a
460 40
2420 20a
360 4
130 1
5.1 0.2
27.1 0.2
61
bDL

3660 1000
3850 500a
2730 210
710 60
4520 50
350 5a
100 1
81
35 1a
4 1a
bDL

Values are means standard error. The subscript letter a indicate differences statistically significant between the groups, in each region at P 0.05.

C.I. group
6160 1930a
2460 340a
2440 170
670 70
4170 40
1605 10a
70 1
91
115 1a
10 2a
51

R.F.B. Serpa et al. / Spectrochimica Acta Part B 61 (2006) 12191223

Acknowledgement
This work was partially supported by Conselho Nacional de
Desenvolvimento Cientifico e Tecnolgico (CNPq) and Light
Synchrotron Brazilian laboratory.
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