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Abstract Human brain (globus pallidus) and liver tissues
were investigated by means of electron microscopy (EM),
Mössbauer spectroscopy (MS) and SQUID magnetometry
techniques. Based on MS measurements, the iron present
was identified to be in the ferritin-like form (61–88%) and
in the form of a low-spin iron species (the balance). Its
overall concentration was estimated as 1.5(3) mg in the
brain and 2.4(5) mg in the liver, per gram of lyophilized
tissue. The average core diameter was determined by EM
measurements to be equal to 7.5(1.3) nm for the liver and
3.3(5) nm for the brain. Magnetization measurements car-
ried out between 5 and 300 K yielded an estimation of an
average blocking temperature, KT
B
L, as equal to 6.7 K and
8.5 K for the liver and the brain, respectively. From the de-
pendence of KT
B
Lon the external magnetic field it was
concluded that the ferritin-like cores in the studied sam-
ples can be regarded as non-interacting particles. Finally,
the uniaxial magnetic anisotropy constant was determined
to be 6×10
3
J/m
3
for the liver and 4×10
4
J/m
3
for the brain.
Key words Human liver · Human brain · Ferritin ·
Electron microscopy · Mössbauer spectroscopy
Introduction
Iron is the most abundant transition metal element in liv-
ing organisms, and it plays a crucial role in many vital
metabolic functions such as oxygen transport and electron
transfer. The positive role it was believed to play in human
health has been recently questioned. Unbound iron is
highly toxic, acting as a catalyst for the production of free
radicals which can ultimately lead to cellular damage. Sev-
eral human diseases, including neurodegenerative Parkin-
son and Alzheimer diseases, have recently been postulated
to be related to the role of iron (Lauffer 1992).
Nature has, however, developed an iron-storage mole-
cule which acts as an internal iron reserve, storing the iron
in a physiologically safe non-toxic form known as ferritin.
Ferritin-like proteins have been found in many diverse or-
ganisms, ranging from primitive bacteria cells to higher or-
ganisms including Homo sapiens. In the latter, ferritin can
be found in various organs such as liver, spleen, heart and
brain, and also in the blood, and its enhanced level is usu-
ally indicative of a disease (Bauminger and Nowik 1998).
Investigation of ferritin is also of interest to the physi-
cist, because ferritin cores, which can accommodate up to
4500 Fe(III) atoms, exhibit superparamagnetic properties
characteristic of magnetic nanoparticles.
In this Letter, magnetic properties of ferritin-like parti-
cles found in a human liver and brain (globus pallidus) are
described and discussed.
Materials and methods
Samples to be investigated were taken from the brain (glo-
bus pallidus) and liver at autopsy using plastic blades. One
brain sample, hereafter referred to as fresh, was sealed in
a lucite container of 1 ml volume and 1.5 cm
2
cross sec-
tion and frozen in dry ice immediately after autopsy. An-
other brain sample and one liver sample were lyophilized
and stored at room temperature.
Three experimental techniques, Mössbauer spectros-
copy (MS), electron microscopy (EM) and SQUID mag-
netometry, were applied in the investigation of the sam-
ples. MS was used to identify what iron species were
present and to estimate their concentration, EM to deter-
mine the size distribution of the ferritin cores, and SQUID
magnetometry to measure magnetic properties of the sam-
ples.
Eur Biophys J (1999) 28: 263–267 © Springer-Verlag 1999
Received: 10 July 1998 / Revised version: 29 September 1998 / Accepted: 9 October 1998
S.M. Dubiel · B. Zablotna-Rypien · J.B. Mackey
J.M. Williams
Magnetic properties of human liver and brain ferritin
BIOPHYSICS LETTER
S.M. Dubiel (½) · B. Zablotna-Rypien
Faculty of Physics and Nuclear Techniques,
The University of Mining and Metallurgy (AGH),
al. Mickiewicza 30, PL-30-059 Kraków, Poland
e-mail: dubiel@novell.ftj.agh.edu.pl
J.B. Mackey · J.M. Williams
Physics Department, University of Sheffield,
Sheffield S3 7RH, UK
Mössbauer spectra were recorded in a transmission
geometry by means of a standard spectrometer on samples
whose mass was ≈200 mg. A 40 mCi source of
57
Co/Rh
kept at room temperature was used to supply the 14.4 keV
gamma rays, which were detected by a proportional coun-
ter. During measurements the samples were kept in a gas-
flow cryostat, and their temperature Twas varied between
80 K and 290 K with an accuracy of 0.1 K. A least-squares
fit to the Mössbauer data yields values for the hyperfine
parameters (the isomer shift, IS, the quadrupole splitting,
QS) as well as the line width,
Γ
, and the line intensities.
TEM micrographs were taken on ferritin particles iso-
lated from the tissue by means of a JEM 1200 ExII elec-
tron microscope operating at 80 keV. Particle size deter-
minations were made by measurement of 60 protein iron-
cores.
Magnetization measurements were carried out on the
lyophilized samples (of mass ≈100 mg) using the MPMS-
5 SQUID magnetometer from Quantum Design. It has a
temperature range of 5–300 K and can produce magnetic
fields up to 5 T. The zero-field-cooled magnetization
curve, M
ZFC
versus T, was measured on the samples that
were initially cooled to 5 K in zero field. Then a constant
field was applied and the magnetization measured in that
field as a function of increasing temperature. The field-
cooled magnetization curve, M
FC
versus T, was then
recorded on cooling the sample down to 5 K in the field.
The fields applied in the present study were between 50 G
and 4000 G for the liver and 50 G and 1400 G for the
brain.
Results and discussion
Mössbauer effect
Figure 1 shows a
57
Fe Mössbauer spectrum recorded at
80 K on a sample of horse spleen ferritin from Sigma,
which was used as a standard. It was fitted in terms of a
doublet whose best-fit spectral parameters are as follows:
IS = 0.45(1) mm/s (relative to that of
α
-Fe), QS = 0.70(1)
mm/s and
Γ
= 0.62 mm/s. Mössbauer spectra of the inves-
tigated samples are presented in Fig. 2. In comparison with
that shown in Fig. 1 they have an additional subspectrum,
so they were therefore successfully fitted in terms of two
doublets. The best-fit spectral parameters thus obtained are
displayed in Table 1. They prove that the investigated sam-
ples contain iron in the form of ferritin (61–88%) and a
low-spin iron (balance) (St. Pierre et al. 1992). The con-
tent of the latter seems to be enhanced by the lyophiliza-
tion.
Knowing the concentration of the iron in the standard
sample, C
0
, its content in the investigated samples, C
x
, was
determined from the formula
C
x
=C
0
(S
x
/S
0
) (1)
where Sstands for the normalized spectral area. The val-
ues obtained can be seen in Table 1 and agree well with
264
Fig. 1
57
Fe Mössbauer spectrum of a horse spleen ferritin from Sig-
ma recorded at 80 K
Fig. 2a–c
57
Fe Mössbauer spectra recorded at 80 K for afresh and
blyophilized sample of brain (globus pallidus) and clyophilized
sample of liver
those available in the literature (Galazka-Friedman and
Friedman 1997).
Electron microscopy
The size distribution of ferritin cores isolated from the liver
tissue is presented in Fig. 3. The average core diameter is
equal to 7.5(1.3) nm, which agrees with estimates found
for ferritin from human spleen, heart and liver (St. Pierre
et al. 1992). However, similar measurements done on the
brain-ferritin cores yielded for the average diameter the
value of 3.3(5) nm (J. Galazka-Friedman, personal com-
munication, 1997). This means that the volume of the liver-
ferritin cores is one order of magnitude larger than that of
the brain-ferritin.
SQUID magnetometry
Examples of typical field-cooled (FC) and zero-field-
cooled (ZFC) magnetization curves obtained are shown in
Fig. 4. Their characteristic features are: (1) a maximum in
the M
ZFC
curve, whose position is usually associated with
the average blocking temperature, KT
B
L(examples of the
maxima recorded for the brain can be seen in Fig. 5) and
(2) a bifurcation of the two curves at T
B
which defines a
point of an irreversibility line between reversible and irre-
versible behaviour. T
B
corresponds to the blocking tem-
perature of the largest particle volume (Mohie-Eldin et al.
1994). It is of interest to study the influence of Bboth on
KT
B
Land on T
B
. According to Luo et al. (1991) and Hanson
et al. (1995), an increase of KT
B
Lwith Bindicates the par-
ticles do not interact with each other. If Bcauses a decrease
of KT
B
L, there is an interaction between the particles.
The data shown in Fig. 6 give evidence, both for the
liver (open symbols) and for the brain (full symbols), that
KT
B
Lincreases with B. This agrees with the expectation that
ferritin cores do not interact (they are encapsulated in a
protein shell). Further evidence to support this conclusion
can be inferred from the ratio between KT
B
Land the block-
ing temperature determined from the Mössbauer spectra,
T
BM
. For a system of non-interacting particles the ratio
T
BM
/KT
B
Lshould be in the range 4–7 (Hanson et al. 1995),
while for strongly interacting particles the ratio should be
close to 1 (Morup et al. 1995). Since the human ferritin
T
BM
values lie in the range 30–38 K (Mann et al. 1987, St.
Pierre et al. 1991), the ratio for the present case is equal to
4.5–5.6 for the liver and to 3.6–4.3 for the brain.
From the present magnetization curves one can also de-
termine the irreversibility line. For this purpose the bifur-
cation temperature, T
B
, has been plotted versus Bin Fig. 7
for the liver and in Fig. 8 for the brain. In both cases, T
B
decreases with B. For an ensemble of superparamagnetic
particles, which should describe the ferritin-like cores, the
behaviour can be theoretically described by the following
formula (Mohie-Eldin et al. 1994):
B=a–bT
B
1/2
(2)
where aand bare constants and T
B
is the blocking temper-
ature.
The data shown in Figs. 7 and 8 were fitted with Eq. (2),
and the best fits are marked by solid lines. The T
B
values
obtained are 18.8 K for the liver and 22.4 K for the brain.
As can be seen, the quality of the fits with this ansatz was
not very good (r
2
= 94.6% for the liver and 91.0% for the
brain). In particular, the curvature of the experimental
points is obviously larger than that predicted by Eq. (2). In
view of this, the data were also fitted with the formula nor-
mally used to describe the irreversibility line for spin-
glasses:
B=c[1–T
B
/T
0
]
φ
/2
(3)
where cis a constant and T
0
is T
B
at B= 0. For
φ
= 3 the
irreversibility line is known as the AT line (de Almeida and
Thouless 1978) and for
φ
= 1 it is called the GT line (Ga-
bay and Toulouse 1981). The use of Eq. (3) to describe our
data seems also to be justified by the fact that for both
classes of materials, i.e. superparamagnetic and spin-glass
265
Sample IS
1
QS
1
Γ
1
A
1
(%)IS
2
QS
2
Γ
2
A
2
(%)C
x
Brain (fr) 0.44 (12) 0.76 (15) 0.54 (3) 88 (7) 0.05 (5) 0.62 (6) 0.22 (3) 12 (3) 0.39 (13)
Brain (ly) 0.51 (5) 0.70 (4) 0.54 (4) 61 (8) 0.10 (1) 0.54 (3) 0.32 (3) 39 (7) 1.5 (3)
Liver (ly) 0.58 (9) 0.66 (1) 0.76 (5) 66 (7) 0.12 (1) 0.64 (4) 0.38 (4) 34 (7) 2.4 (5)
Table 1 The best-fit spectral parameters obtained for fresh (fr) and
lyophilized (ly) samples. IS stands for the isomer shift (relative to that of
α
-Fe), QS is for the quadrupole splitting,
Γ
for the line width
at half maximum, Afor the abundance (%) and C
x
for the iron con-
Fig. 3 Size distribution of ferritin cores isolated from liver tissue
ones, the magnetization curves M
ZFC
versus Tand M
FC
versus Tare virtually identical (see, for comparison, Cham -
berlin et al. 1982). Fitting the present data with Eq. (3) re-
sulted in significantly better fits: see the dotted lines in
Figs. 7 and 8 (r
2
= 98.3% for the liver and 99.6% for the
brain). The zero-field T
B
values obtained in this way are
equal to 25.1 K for the liver and 39.2 K for the brain. The
φ
values are 7.3 for the liver and 11.7 for the brain. It should
be noted that in real spin-glasses the exponent
φ
is usually
larger than 3 (Zieba and Lodziana 1996). It is equal to 7 at
the ferro-para-spin-glass multicritical point, where the fer-
romagnetic interactions set in (Toulouse 1980). The value
of
φ
obtained in the present case suggests that the ferritin-
bound iron atoms behave in an external magnetic field
rather like ferromagnetic spin-glasses and not like super-
paramagnetic particles (the average magnetic moment per
core was estimated as ≈100 µ
B
(Mohie-Eldin et al. 1994).
The dependence of T
B
on Bsuggests that there is a spatial
disorder of the magnetic moments similar to that found in
spin-glasses.
Finally, based on the T
B
values obtained from the
SQUID data and the average core diameters obtained from
the TEM measurements, the uniaxial magnetic anisotropy
constant, K, was determined using the approximation
KV =k
B
T
B
(4)
assuming spherically shaped particles. The values obtained
for Kare 6×10
3
J/m
3
for the liver sample and 4×10
4
J/m
3
for the brain sample.
266
Fig. 4 Examples of magnetization curves recorded with a SQUID
magnetometer for the liver sample in various external magnetic fields
Fig. 5 Examples of the maxima in the M
ZFC
vs. Tcurves for the
brain sample. The curves show the best fits to the data
Fig. 6 The average blocking temperature, KT
B
L, versus the external
magnetic field, B, for the liver (open symbols) and the brain (full sym-
bols). The straight lines show the best fits to the data
Fig. 7 The irreversibility line in the B-T plane for the liver. The
solid line represents the best fit to the data with Eq. (2), while the
dotted line is with Eq. (3)
The enhancement of Kfor smaller particles agrees with
other data (Hanson et al. 1995), and it can be ascribed to a
larger contribution to the total anisotropy from the surface
in the case of the smaller brain-ferritin cores. It is worth
noting that the value of Kobtained presently for the brain
agrees well with that found for amorphous Fe
1–x
C
x
parti-
cles having a similar size (Hanson et al. 1995).
Conclusions
Based on the results presented in this study, the following
conclusions can be drawn:
1. The concentration of iron present in the human brain
(globus pallidus) amounts to 1.5(3) mg and that in the
liver to 2.4(5) mg per gram of lyophilized tissue.
2. About 61–88% of the iron present is in the ferritin-like
form, the rest being a low-spin iron.
3. Ferritin cores found in the liver are one order of magni-
tude larger than those in the brain.
4. The average blocking temperature was determined to be
equal to 6.7 K for the liver and 8.5 K for the brain.
5. The maximum blocking temperature was 25.1 K for the
liver and 39.2 K for the brain.
6. Investigated ferritin-like cores behave like non-interact-
ing particles.
7. The irreversibility line can be better described in terms
of a spin-glass formula with the φexponent character-
istic of ferromagnetic interactions between the spins.
8. The uniaxial magnetic anisotropy constant was deter-
mined as 6×10
3
J/m
3
for the liver and 4×10
4
J/m
3
for the
brain.
Acknowledgements J. Galazka-Friedman and A. Friedman are ac-
knowledged for the kind supply of the samples. In addition, one of
us (S. M. D.) wishes to thank the State Research Committee (KBN),
Warsaw, for financial support.
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Fig. 8 The irreversibility line in the B-T plane for the brain. The
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