ARCHIVES OF

GERONTOLOGY
AND GERIATRICS
Archives of Gerontology and Geriatrics
ELSEVIER 21 (1995) 89-97

AI, Zn, Cu, Mn and Fe levels in brain in

Alzheimer's disease

Erzs6bet Andr~isi*a, l~va Farkas a, Henning Scheibler a,

Antal R6ffy b, L~iszl6 Beztir c
alnstitute of Inorganic and Analytical Chemistry, L. FAtv6s University, 1518 Budapest 112.,
P.O. Box 32., Hungary
bNational Institute of Traumatology, Fiumei f~t 17., 1081 Budapest, Hungary
Clnstitute of General and Analytical Chemistry, Technical University, Gelldrt tdr 4, 1111 Budapest,
Hungary

Received 19 September 1994; revision received 8 December 1994; accepted 30 March 1995

Abstract

The aim of our experiments was to investigate the possible element concentration changes
in Alzheimer's disease. Our project incorporated the determination of the regional distribu-
tion of elements in normal human brain, too. Five elements (AI, Zn, Cu, Mn and Fe) have
been measured in 10 different regions of 20 normal brains (mean age 70 years) and in brain
parts of patients with Alzheimer's disease (9 individuals). Analyses were carried out by induc-
tively coupled plasma atomic emission spectrometry (ICP-AES) and instrumental neutron
activation analysis (INAA) techniques. Analytical precision and accuracy were investigated
by NIST standard reference material. In a comparison between the healthy and Alzheimer
patients' concentration data using statistical treatment these elements showed difference as a
function of the Alzheimer disease (AD).

Keywords: Elements; Normal human brain; Alzheimer's disease; ICP-AES; INAA

l. Introduction

It has been k n o w n for over 100 years that trace elements play an vital role in
metabolism. Some 24 elements are now acknowledged as being essential to life.

* Corresponding author, Tel.: 36 I 2090 555; Fax: 36 1 2090 602.

0167-4943/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved

SSDI 0167-4943(95)00643-Y
90 E. AndrLtsi et al./ Arch. Gerontol. Geriatr. 21 (1995) 89-97

Many conditions have been examined in recent years from a trace element viewpoint.
Much research has been directed to looking at a range of trace elements thought to
play a part in neurological diseases. A recent publication (Ehmann et al., 1986)
summarized the elements that have been implicated in Alzheimer's disease (AD),
with pro and contra literature citations. This study demonstrated imbalances in
8 elements in AD brain compared to age-matched controls.
In our previous work we have analysed element concentrations in 6 brain parts
of 11 men having no primary lesions in their nervous system (age-group 65-75)
(Andr~isi et al., 1990). In this work our goal was to investigate more regions in more
control brains than previously as well as to examine the possible element concentra-
tion changes in histopathologically selected 10 brain regions from 9 patients with
AD. Results obtained for 5 elements (AI, Zn, Cu, Mn and Fe) are presented and
discussed for control and AD subjects.

2. Materials and methods

2.1. Sample collection and treatment

The brains were removed at autopsy, within 48-72 h after death, by means of a
standard procedure. The sampling was carried out by using stainless steel knife. All
containers for the samples were polyethylene. The knife and containers were
thoroughly cleaned in analytical grade nitric acid (soaking for 24 h) and double-
distilled water before use. The leptomeninges, which have been shown to have a high
aluminium concentration (McDermott et al., 1979), were removed from all samples.
The brain's different regions are known to be devoted to different functions,
therefore, bulk analysis is not adequate. Tissue samples were dissected from 10
different regions of both hemispheres.
The samples were conserved in formalin. The formalin blank and the problem of
leaching were examined, too. Additional details of experimental procedure have
been published elsewhere (Andrfisi et al., 1990).
The wet weight was determined and the specimen placed in an oven at 105°C for
36 h reaching constant mass. Sample sizes typically ranged from 10 to 250 mg dried
tissue. The dry mass contents were calculated.
In the case of AD diagnosis was done by clinical and histopathological methods.
AD patients had a history of progressive dementia. Histological diagnosis confirmed
diffuse neurofibrillary tangles (NFTs) and neuritic plaque formation in the
neocortex. Control subjects had no history of dementia or neurological diseases and
no or minimal NFT or neuritic plaque formation. The population consisted of 20
controls and 9 AD subjects. The average age of all patients was 70 years.
Two digestion methods were applied. One of them was carried out in a Parr high-
pressure bomb (sample mass: 100 mg; 2.5 ml 65% nitric acid, 150°C, 2 h). In the sec-
ond case we used microwave heating for sample dissolution (sample mass: 100 mg;
3 m165% nitric acid at 70 psi for 8 min at 100% power and for 12 rain at 80% power).
After cooling the bombs to room temperature, the contents were diluted to 5 ml with
distilled water.
 E. Andr~i et al./Arch. Gerontol. Geriatr. 21 (1995) 89-97 91

2.2. Applied techniques

The dissolved samples were analysed by inductively coupled plasma atomic emis-
sion spectrometry (ICP-AES) on a Labtest Plasmalab ICP spectrometer in argon
plasma (incident power 1.3 kW, radiofrequency 27.12 MHz, observation height
16 ram). Sample introduction was based on flow injection of a 130-#1 sample using
a pneumatically operated injection valve. Background correction and an element-
specific interference correction was applied using a special program handling all the
measurement data, resulting in good reproducibility.
The dried samples were measured directly by instrumental neutron activation
analysis (INAA) in the nuclear research reactor of the Technical University,
Budapest. The samples (100 mg) and the comparator (ruthenium powder) were en-
capsulated in polyethylene vials. The brain samples were irradiated in 2 steps by
thermal neutrons with a flux of 2.10 t2 neutron cm -2 s -1. The 7-spectra were
detected by a Ge(Li) detector and evaluated by a Canberra-80 analyzer connected
to a TPA (PDP 1140 compatible) minicomputer. After correction for background,
peak areas were calculated to determine the concentrations of elements.
A more detailed description of the methods has been provided elsewhere (Andr~isi
et al., 1990). In order to investigate the precision and accuracy obtainable with both
methods, standard solutions (obtained from high purity compounds dissolved in
ultrapure nitric acid) and different biological standard reference materials were anal-
ysed (Andr~isi et al., 1993).

3. Results and discussion

According to our investigations the water content of every specific brain part
differs so much that it was necessary to determine the dry mass content for each nor-
mal and pathological sample (Table 1). The differences in dry mass content between
AD and controls are significant indicating a slightly greater water content in some
AD brain parts.
Various methods have been employed for tissue conservation but care has to be
taken in preservation, as elements may be leached out by the process of conservation
or incorporated into the sample from formalin and this leads to difficulty in compar-
ing results from different studies due to the different methods employed. With regard
to main components (e.g. K, Na) we recognized the leaching problem in formalin
conserved brain parts, therefore in our study conclusions will be drawn only about
the trace elements' alterations depending on disease. From our earlier experiments
it was seen that the leaching of trace elements is insignificant, because these elements
are bound to the proteins and the formalin fixed their structure.
Sample digestion using microwave heating proved to be a rapid method for diges-
tion of brain samples. Using nitric acid and applying our parameters yielded elemen-
tal data which are in good agreement with Parr-bomb digestion values if the
naturally oceuring difference between the subjects is taken into account (Table 2).
INAA and ICP-AES has been employed to determine element levels in human
brain parts. The INAA method is unique because it eliminates the usual problems
92 E. Andrdsi et al./ Arch. Gerontol. Geriatr. 21 (1995) 89-97

Table 1
Dry mass contents of normal and AD brain parts

Brain parts Dry mass* (%)

Normal AD

Ammon's horn 18.31 14.13

Cortex entorhinalis 16.57 16.38
Cortex frontalis parasagittalis 16.19 15.13
Cortex frontalis basalis 16.83 15.05
Area oc¢ipitalis 20.70 17.04
Cortex centralis 17. ! l 17.74
Thalamus 18.66 18.08
Caudatum 16.27 11.83
Putamen 17.25 15.42
Pallidum 23.68 15.45

/'/=9.
*The relative standard deviations were ±2-5%.

(loss and contamination) since there is no sample treatment. The ICP-AES technique
is currently one of the most powerful methods for determination of components of
dissolved samples. Brain parts, of which sufficient quantities were possessed, were
analysed by the above-mentioned independent methods. The elemental concentra-
tions determined by these methods are in a good correlation with one another, if
possible inhomogeneity inside the brain part is taken into account (Table 3).
Unfortunately no certified standard reference material for human brain analysis
exists. The elemental composition of a few animal standard reference materials,
especially Bovine Liver 1577 SRM (NIST) and the human brain is similar. The anal-
ysis of this standard reference material can be used for certification of our data on
human brain parts (LaFleur at al., 1977). Our figures are in good agreement with
certified values (Table 4). There is no certified value for aluminium, but our value

Table 2
Comparison of microwave (M) and Parr-bomb digestion (P) at the analysis of brain parts

Element Caudatum Cortex frontalis basalis

P M P M

Ai 2.2 1.8 2.5 2.6

Cu 23.4 22.7 19.6 19.8
Mn 3.00 2.70 1.25 1.33
Zn 75.5 70.0 60.4 61.9
Fe 542 551 217 203

~g/g dry wt. ICP-AES.

 E. Andr~i et al./ Arch. Gerontol. Geriatr. 21 (1995) 89-97 93
Table 3
Analytical results for 2 brain parts determined by ICP-AES and INAA

Element Pallidum (AD) Ammon's horn (Normal)

ICP-AES INAA ICP-AES INAA

Al 3.5 4. 0.4 3.1 4. 0.5 !.4 4. 0.6 --

Ca 360 4. 20 460 4. 50 330 4. 30 320 4. 30
Cu 31 4. 4 32 4. 3 26 4. 4 22 4. 3
Fe 235 4. 35 213 4. 25 259 4. 30 223 4. 30
Mg 630 4. 70 710 4. 80 790 ± 60 840 4. 90
Mn 1.04. 0.1 1.1 4- 0.1 1.3 4- 0.1 1.1 4. 0.1
Zn 59 4. 6 55 4. 8 61 4. 7 55 4. 8

~gdrywt. n=3.

agrees well with reported aluminium concentrations in this standard (Goode et al.,
1977; McDermott et al., 1978; Markesbery et al., 1981). There is a problem with the
analysis of aluminium because of the environmental contamination, therefore the
highest care is necessary to minimize any contamination, especially at sample collec-
tion and treatment.
Our preliminary results with normal human brain parts showed that the cor-
responding regions from both hemispheres of one human brain revealed similar con-
centrations (And~si et al., 1990). A close relation between the concentrations of
particular elements in the right and left sides of the brain was also shown by other
investigators (H6ck et al., 1975; Duflou et al., 1988). This resemblance of the trace
element distribution in both hemispheres indicates that the significant differences for
the different brain areas is not at all accidental.
A picture of trace element pattern in normal human brain is shown in Table 5.
The values obtained in this study are in fairly good agreement with the currently
obtainable literature, although the data available regarding elemental levels in

Table 4
ICP-AES and INAA results for NBS bovine liver SRM 1577

Element Certified value ICP-AES INAA

Ca 124 4. 6 116 4. 10 n

Cu 193 4- 10 205 4. 10 185 4. 10

Fe 270 4. 20 284 4- 10 315 4. 30
K 9700 4. 600 9500 4. 570 9500 4- 400
Mg 604 4. 9 603 4. 80 605 4. 100
Mn 10.3 4. 1.0 11.5 4- 0.8 9.8 4. 0.7
Na 2430 4- 130 2200 4. 40 2300 4. 100
Zn 130 4. l0 147 4. 5 149 4. 10
Ai /2.2-6.0/ 4.5 4. 1

~tg/g dry wt. n = 3.

94 E. Andrdsi et al./ Arch. Gerontol. Geriatr. 21 (1995) 89-97

Table 5
T r a c e element values in various normal human brain regions

Brain parts AI Zn Fe Cu Mn

A r e a occipitalis 1.9 ± 0.7 75 ± 13 520 ± 60 28 ± 8 2.0 ± 0.5

Caudatum 1.5 ± 0.6 80 ± 7 640 ± 100 37 ± 7 3.8 ± 1.7
Putamen 1.6 ± 0.7 91 ± 20 910 ± 190 42 ± 7 4.0 ± 1.7
Pallidum 2.0 ± 0.7 68 ± 5 830 ± 300 40 ± 10 4.4 ± 0.8
Thalamus 2.0 ± 1.0 66 ± 12 240 ± 40 16 ± 6 1.8 ± 0.7
Cortex centralis 2.0 ± 0.8 102 ± 40 150 ± 30 16 ± 6 1.4 ± 0.6
Ammon's horn 1.5 ± 1.1 86 ± I1 230 ± 100 19 ± 7 1.8 ± 0.8
Cortex entorhinalis 1.5 ± 1.1 54 ± I1 230 ± 100 19 ± 7 1.8 ± 0.8
C o r t e x frontalis parasagittalis 2.0 ± 0.8 47 ± 14 150 ± 30 17 ± 5 1.4 ± 0.5
C o r t e x frontalis basalis 2.0 ± 0.8 47 ± 14 150 ± 30 17 ± 5 1.4 ± 0.5

t~g/g dry wt. n = 20.

human brain is rather scarce (Ehmann et al., 1982; Markesbery et al., 1984; Castillo
et al., 1988; Duflou et al., 1988). The data in Table 5 indicate that the concentrations
of Fe, Cu, Zn, and Mn are consistently higher in basal ganglia than in other cortical
structures. The difference is especially pronounced for Mn and Fe. An enrichment
of Zn was observed in central cortex and in putamen. The lowest Zn concentration
was seen in frontal basal and frontal parasagittal cortex. The largest differences be-
tween the essential trace elements and AI pattern were found especially in frontal
basal and parasagittal cortex. The AI concentration does not at all decrease with
decreasing neuron density, the opposite rather is true. AI has no essential physio-
logical function, but it is known to be toxic. Another interesting finding is that some
regions which can be classified on the basis of their physiological function contained
similar high trace element levels (e.g. extrapyramidal system) suggesting that there

Table 6
Trace element data in different AD human brain regions

Brain parts AI Zn Fe Cu Mn

Area occipitalis 6.8 ± 2.5 52 ± 23 380 ± 130 23 ± 2 2.5 ± 0.6

Caudatum 6.5± 1.5 4 9 ± 11 430± 90 29±3 2.5± 1.3
Putamen 4.9 ± 2.4 43 ± 6 600 ± 250 21 ± 5 2.2 ± 0.7
Pallidum 6.3 ± 3.0 41 ± 4 570 ± 220 27 ± 4 2.1 ± 1.0
Thalamus 4.8 ± 3.0 49 ± 17 310 ± 150 33 ± 18 3.0 ± 2.0
C o r t e x centralis 5.1 ± 2.1 39 ± 9 370 ± 50 24 ± 5 2.1 ± 0.8
Ammon's horn 5.2 ± 2.9 46 ± 8 310 ± 60 20 ± 3 2.7 ± 1.3
Cortex entorhinalis 12.2 ± 8.4 42 ± 7 380 ± 40 24 ± 6 3.1 ± i.6
C o r t e x frontalis parasagittalis 7.5 ± 4.1 46 ± 12 400 ± 30 24 ± 8 2.5 ± 0.9
C o r t e x frontalis basalis 6.7 ± 2.9 43 ± 11 340 ± 60 19 ± 5 2.5 ± 0.9

~g/g dry wt. n = 9.

 E. Andr~i et al./Arch. Gerontol. Geriatr. 21 (1995) 89-97 95

is some relation between the trace element profile of the brain and its function (dif-
ferent metalloprotein enzymes).
Table 6 clearly illustrates that the concentration of Al is consistently higher in AD
specimens than in normal human brain parts. The mean Al concentrations in brain
parts ranged from 1.5 to 2 ~g/g for control and from 4.8 to 12.2 ~,g/g for AD samples.
Our experimental precision was ± 5-10% based on replicate analysis of the same
sample. Statistical analysis revealed significant difference between AD samples and
controls at 99% confidence level.
The elevation of A1 in brain of AD subjects has been a consistent imbalance in
our study, especially in entorhinal and in frontal parasagittal areas. Ehmann et al.
(1986) did not observe an Al imbalance in their study of senile dementia subjects and
controls. Ward and Mason (1987) found Al to be elevated in the hippocampus and
cerebral cortex of AD subjects compared to controls. Aluminium has been found in
increased concentrations in all Alzheimer's disease-affected tissues according to the
results of Krishnan et al. (1988).
The possibility to produce memory and learning impairment by intracranial AI
injection in different animal species, and the increased prevalence of Alzheimer's
disease cases in areas with a high Al level in drinking water (Martyn et al., 1989)
support the assumed relevance of AI accumulation in the human brain for the
dementia of the Alzheimer's type. Recently several studies have been undertaken to
investigate the effects of desferrioxamine in AD. Regarding a study with desferri-
oxamine in AD patients, it appears likely that the removal of AI by desferrioxamine
can be related to a relative reduction in the progression of dementia (Crapper et al.,
1991). Other investigations regarding desferrioxamine should be undertaken, to get
more concrete knowledge about desferrioxamine and its possible relevance concern-
ing therapeutical aspects in this disease.
According to our results Zn is significantly decreased in AD patients compared
to controls in almost every investigated brain parts. The lowest value can be found
in the cortex centralis. These results disagree with those of Ehmann et al. (1986), who
reported elevated Zn in the amygdala and observed no Zn alterations in AD sub-
jects' specimens derived principally form the cerebral cortex. Ward and Mason
(1987) and Constantinidis (1992) reported decreased Zn levels in AD hippocampus
and cerebral cortex.
This decrease of Zn may be caused by the primary amyloid plaques, which disturb
the blood brain barrier, leading to the accumulation of different metals in the
cerebral cortex and to displacement of Zn in some enzymes which become nonfunc-
tional (Constantinidis, 1991, 1992). Therapeutical trials with zinc aspartate during
the timing window (14-67 months), which describe the time between amyloid pla-
ques and amyloid plaque-induced NFTs production, were successful (Constan-
tinidis, 1992). Therefore, further investigations are required to prove the relevance
of Zn regarding to therapeutical aspects in AD.
Our Fe results show that significant concentration changes can be observed in
basal ganglia (decrease) and in other cortical structures (increase). Whether iron in-
itiates neurodegeneration in AD or alterations in Fe metabolism occur generating
free radical species that further impair the cells, is not known yet.
96 E. Andrdsi et al./ Arch. Gerontol. Geriatr. 21 (1995) 89-97

It can be seen from Table 6 that there is a reduction in Cu levels in basal ganglia
and increased level in the other A D brain parts (e.g. in thalamus). The picture o f
these elemental concentration changes is very similar to changes which are shown
by Fe. As far as Cu is concerned there is as yet insufficient knowledge about its
relevance for AD. The concentration change o f Cu may relate to specific disease
processes that occur in AD.
Our Mn data are varied in A D subjects compared to the control brains, too.
Elevated Mn concentration was found in the entorhinal cortex, A m m o n ' s horn,
frontal parasagittal and basal cortices, as well as in thalamus, but diminished Mn
concentration in basal ganglia (especially in pallidum). N o w a d a y s the essential role
of Mn in h u m a n organism is known, but there is no idea what kind o f role it has
in AD.
Our results are encouraging and definitely indicate that there are substantial dif-
ferences between the normal and A D brain regions in elemental concentrations. It
is our intention to extend the present study to more normal and A D h u m a n brains
(fresh or deep-frozen) in order to obtain better baseline values for more elements and
extend the discussion and interpretations o f the present paper.

Acknowledgement

The authors thank the National Scientific F o u n d a t i o n (Hungary) project O T K A

2281 for their financial support.

References

Andr~isi, E., N~idasdi, J., Molwlr, Zs., Bez~ir, L. and Ernyei, L. (1990): Determination of main and trace
element contents in human brain by INAA and ICP-AES methods. Biol. Trace Elem. Res., 26/27,
691-698.
Andr~isi, E., D6zsa, A., Bezdr, L., Ernyei, L. and Moln~ir, Zs. (1993): Characterization of biological RMs
for potential use in human brain analysis. Fresenius J. Anal. Chem., 345, 340-342.
Castillo, J.R., Fernandez, A. and Bona, M.A. (1988): Zinc determination by atomic absorption spectrom-
etry in several types of human biological fluids and tissues. At. Spectroscopy, 9/b, 200-202.
Constantinidis, J. (199I): The hypothesis of zinc deficiencyin the pathogenesis of neurofibrillary tangles.
Med. Hypotheses, 35, 319-323.
Constantinidis, J. (1992): Treatment of Alzheimer's disease by zinc compounds. Drug Dev. Res., 27,
1-14.
Crapper, D.R., McLachlan, D.R., Dalton, A.J. and Kruck, T.P.A. (1991): Intramuscular desferrioxamine
in patients with AD. Lancet, 1, 1034-1038.
Duflou, H., Maenhaut, W. and De Renck, J. (1988): Trace elements in human brain. Regional distribu-
tion and alterations in structures affected by cerebral infarction. In: Trace Element Analytical Chemis-
try in Medicine and Biology, pp. 483-490. Editors: P. Schramel and P. Britter. Walter de Gruyter,
Berlin.
Ehmann, W.D,, Markesbcry, W.R. and Hossain, T.I.M. (1982): Trace elements in human brain tissue by
INAA. J. Radioanal. Chem., 70, 57-65.
Ehmann, W.D., Markesbery, W.R,, Alanddin, M., Hossain, T.I.M. and Brubaker, E.H. (1986): Brain
trace elements in Alzheimer's disease. Nenrotoxicoiogy, 7, 197-206.
Goode, G.C., Herring,ton, J. and Goddard, P.C. (1977): Neutron activation analysis for aluminium in
bone and tissue samples. Radiochem. Radioanal. Lett., 31, 87-94.
 E. Andr6si et ai. / Arch. Gerontol. Geriatr. 21 (1995) 89-97 97

H6ck, A., Demmel, U., Schicha, H., Kasperek, K. and Feinendegen, L.E. (1975): Trace element concen-
tration in human brain. Brain, 98, 0 - 6 4 .
Krishnan, S.S., McLachlan, D.R. and Krishnan, B. (1988): Aluminium toxicity to brain. Sci. Total En-
viron., 71, 59-64.
LaFleur, P.D., Rook, H.L. and Mears, T.W. (1977): Certificate of Analysis, Standard Reference Material
1577, Bovine Liver. National Bureau of Standards, (NIST), Washington.
Markesbery, W.R., Ehmann, W.D., Hossain, T.I.M., Alauddin, M. and Goodin, D.T. (1981): Instrumen-
tal neutron activation analysis of brain aluminium in Alzheimer disease and aging. Ann. Neurol., 10,
511-516.
Markesbery, W.R., Ehmann, W.D., Alauddin, M. and Hossaln, T.I.M. (1984): Brain trace element con-
centrations in aging. Neurobiol. Aging, 5, 119-128.
Martyn, C.N., Barker, D.J.P., Osmond, C., Harris, E.C., Edwarson, J.A. and Lacey, R.F. (1989):
Geographical relation between Alzheimer's disease and aluminium in drinking water. Lancet, 1,
59-62.
McDermott, J.R., Smith, A.I., Ward, M.K., Parkinson, I.S. and Kerr, D.N.S. (1978): Brain aluminium
concentration in dialysis encephalopathy. Lancet, 1,901-904.
McDermott, J.R., Smith, A.I., Iqbal, K. and Wisniewski, H.M. (1979): Brain aluminium in aging and
Aizheimer disease. Neurology, 29, 809-814.
Ward, N.L and Mason, J.A. (1987): Neutron activation analysis for identifying elemental status in
Alzheimer's disease. J. Radioanal. Nucl. Chem., 113, 515-526.