Pharmacology & Pharmacy, 2011, 2, 116-121
doi:10.4236/pp.2011.23015 Published Online July 2011 (http://www.scirp.org/journal/pp)
Morphological and Functional Alterations in
Human Red Blood Cells Treated with Titanium
Citrate
Gugliotta Tiziana, De Luca Grazia, Romano Pietro, Rigano Caterina, Romano Orazio,
Scuteri Adriana, Romano Leonardo
Department of Scienze della Vita “M. Malpighi”, Sezione di Fisiologia generale e Farmacologia, Facoltà di Scienze MM.FF.NN.,
Università degli Studi di Messina, Messina, Italy.
Received February 24th, 2011; revised May 2nd, 2011; accepted June 10th, 2011.
ABSTRACT
The morphological and functional effects of titanium (Ti) citrate on human erythrocytes were studied by scanning elec-
tron microscope (SEM), sulphate uptake via band 3 protein and by determining the reduced and oxidised glutathione
(GSH and GSSG, respectively) concentrations. The rate constant for sulphate uptake was significantly lower after Ti
citrate treatment. Ti citrate (0.001 and 0.0025 mM) significantly decreased erythrocyte GSH and increa sed GSSG con-
centrations. At 0.005 mM Ti citrate, the intracellular GSH could not be tested due to significant cellular damag e. SEM
of erythrocytes treated with 0.001 mM and 0.0025 mM Ti citrate showed structural membrane defects but almost nor-
mal cellular diameters. At even higher Ti citrate concen trations (0.005 mM), erythrocytes showed obvious morphologi-
cal alteration and shape changes compromising the cells physiological functions. In conclusion, although the Ti con-
centrations used in our experiments are physiologically high, the cumulative effect of prolonged exposure to much
lower doses of Ti, as might occur during total hip replacement, should be considered for further experimental testing.
Keywords: A nion Transport, Band 3 Protein, Erythrocytes, E ry throcyte Membran e, GSH, Titanium Citrate
1. Introduction
Titanium (Ti) is largely present in nature in the form of
oxides and it is contained in different minerals. It is the
ninth element in order of diffusion and the fourth in
order of abundance among the metals, preceded only
by aluminum, iron and magnesium. Modern extraction
technologies have allowed the use of Ti both in the
aereonautic-spatial industry as well as some medical
fields, particularly orthopedics, cardio-vascular surgery
and dental surgery. Among the many qualities that
make Ti useful for implantation, are its long-term hard-
ness and biocompatibility. The success of using pure Ti
in medicine is due to its unique physical and chemical
characteristics, not readily found in other metals, such
as its X-ray transparency that helps in diagnosing the
state of prosthesis conservation.
Ti compounds are currently the subject of much bio-
logical research [1,2]. A recent study has investigated
Ti compounds as anticancer agents because they are
easily disseminated throughout the body [3]. One the
few studies showed that osteoblast-like cells respond to
titanium particles through increased expression of the
proinflammatory cytokine interleukin-6 in a process re-
quiring phagocytosis and intracellular signaling path-
ways. Other than this specific research, very limited
data are available on the toxic effects of soluble Ti (IV)
complexes in erythrocytes and in particular the effect
on band 3 protein. Band 3 protein is the most abundant
membrane protein in human erythrocytes, being pre-
sent at approximately 1 million copies per cell [4], and
it facilitates the electroneutral exchange of Cl– and
3
HCO

across the membrane.
Three anion exchanger isoforms are known, AE1,
AE2 and AE3, which differ in their tissue expression.
AE1 is found in erythrocytes and in the kidney, AE2 is
found in a wide variety of tissue and AE3 is found in
the brain, the retina and the heart. All the anion ex-
changer family consist of two domains, an N-terminal
cytoplasmic domain that contains binding sites for
glycolytic enzymes and haemoglobin and a C-terminal

Morphological and Functional Alterations in Human Red Blood Cells Treated with Titanium Citrate 117
membrane domain [5,6]. The membrane domain is
highly conserved, it spans the lipid bilayer 12 - 14
times and mediates anion transport. The cytoplasmic
domain is anchored to the cytoskeleton and plays a
structural role by connecting the cytoskeleton to the
membrane [7,8].
Band 3 protein is one of the main phosphorylated
membrane proteins. It is phosphorylated mainly at
three sites in the cytoplasmic domain, the most impor-
tant being tyrosine-8, followed by tyrosine-21 and ty-
rosine-46 [9]. Band 3 protein phosphorylation is ac-
companied by rigidification of the membrane skeleton,
suggesting the visco-elastic properties of human eryth-
rocytes may be regulated by band 3 tyrosine phos-
phorylation [10].
The aim of our study was, therefore, to expose hu-
man erythrocytes to different concentrations of Ti cit-
rate (IV) in order to study: 1) its effects on the mecha-
nisms of anionic transport mediated by band 3 protein,
2) its oxidative effects by the determination of -SH
groups and intracellular concentrations of GSH and
GSSG and 3) its action on the erythrocyte membrane
by scanning electron microscopy (SEM).
2. Materials and Methods
2.1. Erythrocyte Preparation
Human blood was obtained, after informed consent, from
30 healthy volunteers. The blood was collected in heap-
rinnized tubes, washed in 150 mM NaCl, 25 mM HEPES,
pH 7.40 and centrifuged three times for 5 min at 3000
rpm. At each step the buffy coat was carefully removed
and the cells were suspended at 3% hematocrit and in-
cubated for 1 h at 25˚C in the following medium: 140
mM choline chloride, 10 mM NaCl, 25 mM HEPES, 10
mM glucose, pH 7.4, ± increasing concentrations of Ti
citrate (0, 0.0005, 0.001, 0.0025 and 0.005 mM). After
incubation, the suspensions were centrifuged and the ery-
throcytes were divided into three aliquots. These were
used to study sulphate kinetics, to indirectly verify the
oxidative effects exercised by Ti citrate on the -SH
groups of the membrane proteins, for measuring the in-
tracellular concentrations of GSH and GSSG and to ob-
serve the red blood cell shape by SEM.
Ti citrate (IV) was obtained by mixing Ti III chloride
with sodium citrate in the ratio 1:1.2, at pH 3. The Ti
citrate solution was used after verifying the absence of
precipitates and after solutions become completely clear,
to avoid interference with our experimental protocol [11-
13].
2.2. Sulphate Transport Measurement
For kinetic studies, the erythrocytes were resuspended in
3% hematocrit and incubated at 25˚C in the following
medium: 115 mM Na2SO4, 5 mM Na3(C6H5O7)2H2O, 5
mM KCl, 25 mM HEPES, 5 mM glucose  increasing
concentrations of Ti citrate (0, 0.001, 0.0025 and 0.005
mM), pH 7.40. At specified intervals, 5 ml samples of
the suspension were removed and added to a test tube
containing 5 M 4,4’-diisothiocyanato-stilbene-2,2’-di-
sulfonate (DIDS) stopping medium and incubated on ice.
DIDS binds specifically and irreversibly to the band 3
protein and inhibits sulphate transport in human red
blood cells. In the same experiments, DIDS was added to
the red blood cell suspension to give a final concentra-
tion of 5 M.
After withdrawal of the last sample, the erythrocytes
were washed three times in a sulphate-free medium at
0˚C to remove extracellular sulphate. Cells were then
lysed with distilled water and trichloroacetic acid. The
membranes were removed by centrifugation and sulphate
ions in the supernatant were precipitated by adding glyc-
erol and distilled water (1:1), 4 mM NaCl and HCl (37%)
(12:1), and 124 mM BaCl2 dihydrate, to obtain a homo-
geneous barium sulphate precipitate. The intracellular
sulphate concentration was measured by atomic absorp-
tion spectrophotometry at 425 nm. Using a standard cur-
ve, obtained by precipitating known sulphate concentra-
tions, we converted the absorption to mM intracellular
sulphate and calculated the rate constant in min-1 by a
non-linear, least square, curve-fitting procedure applying
the following equation:
C(t) = C∞(1 – e–rt) + C0
where C0, Ct and C represent the intracellular sulphate
concentrations measured at times 0, t and  [14,15], e
indicates nepero number ( 2.7182818) and r is a constant.
2.3. GSH Measurement
The GSH concentration was measured in erythrocytes
before and after treatment with 0.001 and 0.0025 mM Ti
citrate using an immunodiagnostic assay [16], intended
for the quantitative determination of glutathione in EDTA-
blood. At 0.005 mM Ti citrate, the intracellular GSH was
not possible to test because of damage to the erythrocyte
membrane.
In this assay, the sample was treated with a dilute so-
lution of Ti citrate and divided into two aliquots: 1) The
reduced fraction was measured by adding 50 l of the
diluted sample, 100 l reaction buffer and 100 l deri-
vatization solution. After incubation for 20 min at 60˚C,
during which time GSH was converted to a fluorescent
product, 100 l of the precipitation solution were added
to remove higher molecular weight substances. The sam-
ples were precipitated for 10 min at 2˚C - 8˚C and centri-
fuged for 10 min at 6000 rpm, then 200 l of supernatant

118 Morphological and Functional Alterations in Human Red Blood Cells Treated with Titanium Citrate
were added to 200 l of the reaction buffer in autosam-
pler vials. 2) The total glutathione was measured by
adding 50 l of the diluted sample, 20 l of the reduction
solution, 100 l of the internal standard and 100 l of the
derivatization solution. The sample was then handled
like the reduced fraction. After that, 20 l of the super-
natant were injected into the HPLC system. The separa-
tion by HPLC followed an isocratic method at 30˚C us-
ing a reversed-phase column in two runs.
The chromatograms were scanned by a fluorescence
detector and concentrations were calculated by integra-
tion of the peak height by the external standard method
for the reduced fraction and the internal standard method
for the total glutathione fraction. The amount of oxidised
glutathione was calculated by subtraction of:
glutathione total
– glutathione reduced
2.4. Preparation of Erythrocyte Membrane,
Isolation of Band 3 Protein and
Determination of Sulphydryl Groups
Erythrocytes (with/without Ti treatment) were washed
with an isotonic solution (150 mM NaCl, 25 mM HEPES,
pH 7.40) and hemolysed by adding 20 volumes of cold
hypotonic buffer (5 mM HEPES, pH 7.40). Membranes
were obtained by centrifugation at 20,000 g for 30 min at
4˚C. The process was repeated with the same hypotonic
buffer until the red blood cell membranes were almost
free of hemoglobin [17]. One volume of red blood cell
membrane was then incubated with nine volumes of 0.1
N NaOH for 30 min at 0˚C in the presence of 0.2 mM
DTT (dithiothreitol) and 20 g/ml PMSF (phenylmethyl-
sulfonyl fluoride). After incubation, samples were cen-
trifuged at 20,000 g for 30 min at 4˚C.
The pellet containing band 3 protein was washed three
times with 5 mM HEPES, pH 7.40, and used for deter-
mination of sulphydryl groups. Membrane with/without
Ti treatment and containing band 3 protein was incu-
bated with 0.3 ml of 20% SDS (Sodium Dodecyl Sul-
phate) and 2.8 ml of 100 mM sodium phosphate, pH 8,
for 25 min at 37˚C. The pellet suspension was further
incubated with 0.1 ml of 10 mM DTNB (2-nitrobenzoic
acid) in 100 mM sodium phoshate, pH 8, for 20 min at
37˚C. The sulphydryl group concentration was measured
by atomic absorption spectrophotometry at 412 nm [18].
A standard curve was employed to calculate the concen-
tration of thiol groups.
2.5. Scanning Electron Microscopy and Human
Erythrocytes
The sample preparation technique for electron micros-
copy involved: fixation, dehydration, assembly, covering
with gold and observation. To analyse the effect of Ti
citrate on the morphology of human red blood cells, the
samples were incubated in the presence or absence of
increasing concentrations of Ti at 25˚C for 1h. Samples
were then washed with physiological solution, 166 mM
NaCl, and fixed overnight at 5˚C by adding one drop of
each sample to plastic tubes containing 1 ml of 4% glu-
taraldehyde in Sorensen's phosphate buffer 0.1 M to pH
7.4. Samples were washed three times in the same buffer
for 30 min and then exposed to increasing concentrations
(30%, 50%, 70%, 90% and 95%) of ethanol for about 30
min each.
The process of dehydration was carried out with liquid
carbon dioxide until the critical point was reached. Then
samples were assembled on a particular type of glass
using conductive silver paste and covered with a thin
layer of gold (200 - 300 A) in appropriate sputtering. The
samples were then ready for observation by SEM.
3. Statistical Analysis
In order to test the existence of homogenity between un-
treated (control) and Ti treated cells, we applied non-
parametric permutation tests. This procedure is more
conservative than homologue parametric tests for small
samples [19].
4. Results
The experiments were carried out in order to highlight
not only possible modifications of sulphate transport me-
diated by band 3 protein, but also variations in the levels
of GSH, the shape of human red blood cell membranes
and oxidation of -SH groups. We used low concentra-
tions (0.0005 and 0.001 mM) and high concentrations
(0.0025 and 0.005 mM) of Ti citrate. Sulphate influx in
the control red blood cells and in cells treated with in-
creasing concentrations of Ti citrate are reported in Fig-
ure 1.
The rate constant of the Ti citrate (0.001 mM) treated
sample was 0.026 ± 0.001 × min–1 (Table 1), compared
to 0.039 ± 0.001 × min–1 in the control sample, a reduc-
tion of 33% relative to the control. In Figure 1, the curve
(■) represents the cells treated with an intermediate con-
centration (0.0025 mM) of Ti citrate. The last curve
shows an increase in the time necessary to reach the satu-
ration equilibrium of sulphate.
In Figure 1, the graph (▲) represents the sulphate in-
flux in human red blood cells treated with 0.005 mM of
Ti citrate. It shows a remarkable difference in the kinetic
profile. In fact it can be seen that sulphate saturation
equilibrium was reached suddenly, because band 3 pro-
tein lost its function of anion transporter due to damage
by the titanium.
Figure 2 shows a progressive decrease in the percen-
tage of sulphydryl groups in band 3 protein with in-
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Morphological and Functional Alterations in Human Red Blood Cells Treated with Titanium Citrate 119
Figure 1. Sulphate influx in human erythrocytes measured
in the absence of Ti citrate () or in the presence of 0.001
mM (), 0.0025 mM () or 0.005 mM (▲) Ti citrate. On
the abscissas, the time of collection of the cellular suspen-
sion is reported in minutes. On the ordinates, the corre-
sponding concentrations of the intracellular ion sulphate
are given, expressed in mM × 10–2. The experimental condi-
tions were 25˚C and pH 7.40. Data are reported with S.D.
(standard deviation).
Table 1. Rate constants and percetual inhibition of sulphate
uptake.
Erythrocytes Rate constants × min–1 % of inhibition
Control 0.039 ± 0.001
+ 0.001 mM Ti citrate 0.026 ± 0.001 33.33
+ 0.0025 mM Ti citrate 0.016 ± 0.001 58.97
+ 0.005 mM Ti citrate / /
Figure 2. Reduction of sulphydryl groups of band 3 protein
in erythrocytes treated and untreated with different Ti
concentrations (0, 0.001 and 0.0025 mM). Values are given
as means ± S.D. (standard deviation).
creaseing Ti concentration in treated erythrocytes. The
decrease of -SH group content, observed in treated
erythrocytes, might be the consequence of peroxidation
and oxidation processes elicited by free radical activity.
As shown in Table 2, the GSH concentration in hu-
man red blood cells was reduced after treatment with Ti
citrate (0.001 and 0.0025 mM), as compared to controls.
Table 2. Gsh and gssg concentrations in erythrocytes treated
with Ti.
Control
+ 0.001 mM Ti
citrate
+ 0.0025 mM Ti
citrate
GSH (mol/l)735.26  145697.29  103 602.70  128
GSSG (mol/l) 270.66  36287.89  32 292.49  19
GSH/GSSG ratio2.71  1.2 2.42  1.2 2.06  1.1
Figure 3. Effects of Ti citrate on the morphology of human
erythrocytes. Human erythrocytes observed by SEM: (a)
untreated erythrocytes, 1700x, (b) erythrocytes treated with
0.0005 mM of Ti citrate, 10000x, (c) erythrocytes treated
with 0.001 mM of Ti citrate, 15000x, (d) erythrocytes
treated with 0.0025 mM of Ti citrate, 10000x, (e) erythro-
cytes treated with 0.005 mM of Ti citrate, 10000x.
Intracellular GSH was not determined at 0.005 mM Ti
citrate due to major alterations in the erythrocyte mem-
brane
The oxidative effect highlighted by reduced concen-
trations of GSH, and indirectly the kinetic study of the
sulphate ion exchange, in human red blood cells Ti cit-
rate is corroborated by their morphology as seen by SEM.
At Ti concentrations of 0.0005 mM and 0.001 mM SEM

120 Morphological and Functional Alterations in Human Red Blood Cells Treated with Titanium Citrate
revealed small membrane alterations but an almost nor-
mal cellular diameter (Figures 3(b) and (c)), compared
with the control (Figure 3(a)). Increasing concentrations
of Ti citrate (0.0025 and 0.005 mM) (Figures 3(d) and
(e)) showed increased morphological damage of the
erythrocyte population.
5. Discussions
Band 3 protein mediates anion-exchange and acid-base
equilibrium through the red blood cell membrane. It is an
integral membrane protein crossing the bilayer lipid
membrane many times. Because of its position, the pro-
tein is continuously damaged by chemical agents and
drugs circulating in the blood flow. Previous studies re-
ported that some metals enhance degradation of band 3
protein after treatment with chemical substances likely to
affect the sulphate permeability and modulate anion in-
flux through the erythrocyte membranes [20].
In this set of experiments, sulphate uptake measured in
control erythrocytes increased steeply at the intial stage
reaching equilibrium in 30 minutes. This process was
much slower across the membrane of Ti citrate treated
cells. The decrease in the rate constant of influx in treated
cells compared to control cells was more prominent after
treatment with 0.0025 mM of Ti citrate (Figure 1). Ta-
ble 1 indicates significant inhibition (33% and 58%) of
rate constant sulphate influx across the red cell mem-
brane after Ti citrate treatment. The structural integrity of
band 3 protein has a close relationship with the func-
tional aspects of this anion channel protein [15], and
these results suggest a conformational change in the an-
ion channel protein during Ti treatment making the
transport sites more vulnerable to modification (Figures
3(a-e)).
Marked degradation of membranes and band 3 protein
resulted from using 0.005 mM of Ti citrate. In this case it
was not possible to measure the rate constant in treated
cells. This probably correlated with the gradual reduction
in the percentage of sulphydryl groups (Figure 2) due to
oxidative damage in the membrane because of a signifi-
cant reduction in GSH (735 mol/l in control cells and
287 mol/l in Ti treated cells, Table 2). The GSH reduc-
tion measured in Ti-treated erythrocytes may have al-
tered the properties of hemoglobin and increased its ten-
dency to aggregate, consequently modifying the hemo-
globin-band 3 protein interaction [21]. In contrast, the
binding of the chemical inhibitor DIDS at the anion
binding site of band 3 protein is known to block the up-
take of anion across the cell membrane. However, this
does not deform the membrane. In fact, the GSH defi-
ciency (Table 2) measured in Ti-treated erythrocytes al-
tered the properties of haemoglobin and increased its
tendency to aggregate, with consequent modification of
the hemoglobin-band 3 protein interaction [21,22].
SEM of erythrocytes treated with increasing concen-
trations of Ti revealed significant morphological differ-
ences compared to untreated erythrocytes. In Figure 3,
only a few of the erythrocytes exposed to lower concen-
trations of Ti citrate (0.0005 and 0.001 mM) seem slight-
ly deformed in shape compared to control cells. At high-
er concentrations of Ti citrate (0.0025 mM), most of the
erythrocytes showed morphological alterations. With
0.005 mM of Ti citrate the erythrocyte membranes were
damaged and cells appeared smaller and more distorted.
This is probably due to insertion of Ti citrate into the
lipid bilayer of the erythrocyte membrane.
In conclusion, although the Ti concentrations used in
these experiments were unphysiologically high compared
to Ti circulating, for example, after total hip replacement,
our findings warrant examination of the effect of long-
term exposure of erythrocytes to Ti and also check whe-
ther the structural alterations induced by titanium citrate
to human red blood cells can be extended to other cells
affecting their functions.
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