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ORIGINAL SCIENTIFIC PAPER
Croat. Chem. Acta , 91()
Published online:
Electrochemical Determination of Metallothioneins
by the Modified Brdička Procedure as an Analytical
Tool in Biomonitoring Studies
Tatjana Mijošek,* Marijana Erk, Vlatka Filipović Marijić, Nesrete Krasnići, Zrinka Dragun, Dušica Ivanković
HR-10000 Zagreb, Croatia
* Corresponding author’s e-mail address:
PROCEEDIN G OF THE 5TH DA Y OF ELECTROCHEMIS TRY AND 8TH ISE SSRSE, 25 MAY 2018, ZAGREB, CROATIA
Abstract: alysis
–1 –1
measurements, in both standard sample and sa-
-saving
improvement is especially use
Keywords: ring
INTRODUCTION
ETALLOTHIONEINS (MTs) are the family of inducible
metal-binding proteins which have important roles
in the homeostasis of essential trace metals (Cu and Zn)
and/or sequestration of toxic metals (e.g., Ag, Cd and Hg),
as well as in the protection against oxidative damage.[1,2]
They are characterized by low molecular mass, lack of
aromatic amino acids and high cysteine content. Increased
MTs reduce toxic effects caused by metals and at cellular
level are one of the first detectable signs of the occurrence
of elevated metal levels. Therefore, MTs are often used in
biomonitoring studies as biomarkers of metal exposure.[2,3]
Analytical methods used for MT determination cover
a broad range of techniques including direct detection of
MTs (immunochemistry, mass spectrometry and electro-
chemistry) and indirect detection of MTs (saturation assays
with Hg, Cd or Ag, and spectrometric methods via metal
detection in proteomics).[4,5] A method for polarographic
determination of proteins containing thiol groups in ammo-
nia buffered cobalt(III) solution was first introduced by
Brdička.[6] Since then, the procedure has been modified by
Paleček and Pechan,[7] Kehr,[8 ] Olafson and Sim,[9] Thomp-
son and Cosson,[10] and Olsson and Haux.[11] Therefore, the
method used today is commonly known as modified
Brdička reaction. Main improvements from the original
procedure include application of pulse techniques and
hanging mercury electrode instead of the dropping one,[9]
which resulted in better resolution and more reliable quan-
tification. The exact mechanism of the reaction was eluci-
dated by Raspor.[12] Quantification of MTs in the reaction is
based on linear relationship between the catalytic hydro-
gen evolution signal and protein concentration.[12] Besides,
it was discovered that the height of the catalytic hydrogen
evolution signal was markedly affected by the temperature
of the analysed solution and by the concentration of
Co(NH3)6Cl3 (depolarizer). The dependence of this signal
height on the temperature indicates that it is not a diffu-
sion, but an adsorption controlled reaction.[13 ] The concen-
tration range of the unknown analyte isolated from the
biological material has to be in the range of the calibration
curve obtained by using commercially available rabbit liver
M
2 T et al.: iomonitoring
Croat. Chem. Acta , 91()
MT as a calibrant.[12,13] This MT calibration is linear in a very
narrow and low range and therefore, biological samples
containing MTs usually have to be diluted prior to the
analysis.
The duration of the measurement of a single sample
was quite time-consuming using the previously defined
measurement parameters[13] for the modified Brdička pro-
cedure. Since the duration of an analytical measurement is
especially important when applied as a routine in biomoni-
toring studies which require analyses of numerous samples,
we modified the duration of one step potential and conse-
quently the scan rate to stipulate if the measurement pro-
cedure would still be reliable but faster. Hence, our main
goals were: 1) to compare MT levels in fish tissues deter-
mined by differential pulse voltammetry (DPV) using two
sets of different measurement parameters and 2) to evalu-
ate the application of the faster modified Brdička reaction
as a sensitive electrochemical method in biomonitoring
studies of metal exposure.
EXPERIMENTAL
Sampling Procedure
Fish sampling was performed by electro fishing, according
to the Croatian standard HRN EN 14011.[14] European chub
(Squalius cephalus Linnaeus, 1758) were sampled in
lowland Sava River and 6 specimens were used for MT
analysis, while brown trout (Salmo trutta Linnaeus, 1758)
were sampled in the karst Krka River in Croatia and 4
specimens were used for MT analysis. Captured fish were
kept alive in an opaque plastic tank with aerated river water
until further processing in the laboratory. Individual fish
were anesthetized with tricaine methane sulphonate (MS
222, Sigma Aldrich, USA) and sacrificed. Liver, gills and the
intestine were dissected, weighed and stored at −80 °C until
further analyses.
Homogenization and Preparation of
Cytosolic Fractions of Fish Liver, Gills
and Intestine
Samples of the fish tissues were cut in small pieces and di-
luted 6 times with cooled homogenization buffer. The ho-
mogenizing buffer used for liver and gills homogenization
contained 100 mM Tris-HCl/base (Merck, Germany, pH 8.1
at 4 °C) supplemented with 1 mM DTT (Sigma, USA) as a
reducing agent. Intestinal tissues were homogenized in ho-
mogenizing buffer containing 100 mM Tris-HCl/base
(Merck, Germany, pH 8.1 at 4 °C) supplemented with 1 mM
DTT (Sigma, USA) as a reducing agent, 0.5 mM PMSF
(Sigma, USA) and 0.006 mM leupeptin (Sigma, USA) as pro-
tease inhibitors. Fish tissues were homogenized by 10
strokes of Potter-Elvehjem homogenizer (Glas-Col, USA) in
an ice cooled tube at 6000 rpm. Obtained homogenates
were centrifuged in the Avanti J-E centrifuge (Beckman
Coulter, USA) at 50,000×g for 2 h at 4 °C. Resulting super-
natants (S50), representing the water soluble tissue frac-
tions (cytosol) were separated and stored at −80 °C for
subsequent analyses.
Heat-Treatment of the Cytosolic
Fractions
Heat-treatment denatures high molecular mass cytosolic
proteins, which would otherwise interfere with the electro-
chemical MT determination.[15] The cytosolic S50 fractions
were 10 times diluted with 0.9 % NaCl (Suprapur, Merck)
and then heat-treated at 85 °C for 10 min in the Dri Block
(Techne, UK). This dilution of S50 fraction with NaCl brings
two benefits for analytical determination of MT – it reduces
MT co-precipitation with high molecular weight proteins
and renders a further sample dilution before the electro-
chemical analysis almost unnecessary.[15] Afterwards, sam-
ples were placed on ice for 30 min at 4 °C and then
centrifuged at 10,000×g for 15 min at 4 °C to allow the co-
agulated proteins to precipitate in Biofuge Fresco centri-
fuge (Kendro, USA) to obtain MT rich fraction. The resulting
heat treated supernatant (HT S50) was stored at –80 °C
until further analyses.
Determination of MT Concentrations
MT concentrations were measured in HT S50 by DPV follow-
ing the modified Brdička procedure.[12,13] Analyses were per-
formed on 797 VA Computrace (Metrohm, Switzerland) with
a three-electrode system (hanging mercury drop electrode,
HMDE, as a working electrode; an Ag/AgCl/saturated KCl ref-
erence electrode; a platinum counter electrode). Measure-
ments were done in 10 mL of electrolyte solution containing
5 mL of 2 M NH
4Cl/NH4OH and 5 mL of 1.2 × 10
–3 M Co
(NH3)6Cl3, pH 9.5 which was thermostated to 20 °C and
purged with the pure nitrogen. Measurement parameters
for DPV are presented in Table 1. MT concentrations were
derived from the calibration straight line, constructed by
using the commercially available standard rabbit liver MT-
2 (Enzo Life Sciences, USA). Standard MT-2 stock solution
was prepared by dissolving 1 g of lyophilised powder in
1 mL of 0.25 M NaCl. Aliquot of 20 µL of 1 mg mL–1 stock
solution was further diluted in 1000 µL of H2O to obtain
19.6 µg mL–1 standard MT-2 working solution.
Calculations and Statistical Analyses
Basic calculations were performed using Microsoft Office
Excel 2007, while statistical analyses were performed using
SigmaPlot 11.0 (Systat Software, USA). Correlation be-
tween two parameter settings applied for the MT measure-
ment in fish tissues was calculated by Pearson correlation
coefficient, and coefficient of determination was calculated
T et al.: iomonitoring 3
Croat. Chem. Acta , 91()
as R2. Variability in MT concentrations of one sample meas-
ured by two parameter settings was calculated as the ratio
of values obtained by two methods and expressed as per-
centage. Measurement accuracy for both methods was cal-
culated in a following way: (cMTaverage – cMTstd) / cMTstd × 100;
precision was expressed as the relative standard deviation
(RSD) and calcula ted in a fo llowing way: SD / cMTaverage × 100;
and recovery (or trueness) was calculated as cMTaverage /
cMTstd × 100.
RESULTS AND DISCUSSION
Calibration Curves
Calibration curves were constructed using MT-2 rabbit liver
standard for both parameter settings, previously used and
explained by Raspor et al.[13] and novel with the modified
instrumental measuring conditions applied in this study.
Linear calibration curves wer e obtaine d by four additions of
standard MT-2 working solution (γMT = 19.6 µg mL –1): 1st ad-
dition - concentration in the cell was 0.04 µg mL–1, 2nd addi-
tion - concentration in the cell was 0.08 µg mL
–1, 3rd
addition - concentration in the cell was 0.12 µg mL–1 and 4th
addition - concentration in the cell was 0.16 µg mL-1. Four
voltammograms were recorded for each addition of stand-
ard MT-2 working solution and the mean value of the MT
peak current at these 4 voltammograms represented one
point of the calibration line (Figs. 1, 2). Voltammograms ob-
tained by the addition of MT-2 standard in several steps
represented the characteristic responses for the mecha-
nism of the modified Brdička reaction explained in detail by
Raspor.[12] In brief, first reduction signal is observed at peak
potential Ep = –1.28 V and it represents the reduction of
Co(III) complex to Co(II). [12] If MTs are present, there is a
reaction between the SH-groups of the proteins and Co(II)
complex and the reduction of this RS2Co complex can be
seen at Ep = 1.35 V.[12] The protons liberated in this reaction
get reduced at Ep = –1.48 V and this step represent the cat-
alytic hydrogen wave which is proportional to the MTs con-
centrations and used for the construction of the calibration
curve and MTs quantification[12] (Figs. 1, 2). It is clearly seen
that with the addition of larger amounts of MTs, the peak
of Co(II) at Ep = –1.28 V decreases because of the formation
of protein complex with MT, while the peak of the RS2Co
comp lex and MT peak inc rease propor tionall y to the MT ad-
ditions [12] (Figs. 1, 2).
Obtained voltammograms and linear calibration
curves are shown in Fig. 1 (duration of one step potential of
0.5 s and scan rate 0.0052 V s–1) and Fig. 2 (duration of one
step potential of 0.2 s and scan rate 0.013 V s–1). The ob-
tained slopes of the calibration straight lines were repeata-
ble and for the operating parameters used in the modified
Brdička procedure by Raspor et al.[13] (duration of one step
potential of 0.5 s), the slope was –5.353 × 10–4, while the
slope obtained with new operating parameters (duration of
one step potential of 0.2 s) was –3.658 × 10–4. In order to
evaluate measurement accuracy and precision, 19.6 µg mL–1
MT-2 rabbit liver standard was added as a sample in the
electrolyte solution and its concentration was measured re-
peatedly twelve times and compared by both methods. For
applied parameter settings using 0.0052 V s–1 and 0.013 V s–1
as a scan rate accuracy was -0.12 % and –2.0 %, RSD was
1.89 % and 1.93 %, while recovery was 99.8 % and 98.0 %,
respectively (Table 2). Therefore, both measurement con-
ditions meet the acceptance criteria set for precision and
accuracy (< 10 %), so presented novel parameter setting of
the modified Brdička procedure has high potential to be ap-
plied in the biomonitoring studies. The main advantage of
the lower scan rate applied in a new instrumental setting is
faster measurement procedure for the MT quantification in
biological samples, giving the increase in the sample
throughput.
Fish Samples
Both DPV parameter settings (scan rate 0.0052 V s
–1 and
0.013 V s–1) were applied for the measurement of unknown
MT concentrations in biological samples, i.e. fish liver, gills
and the intestine. In the biomonitoring studies several fish
organs such as gills, liver and intestine are often used as
target bioindicator organs and MT levels measured in cell
cytosols represent an early warning sign of metal exposure
in the environment.[ 16–20] In the present study, MT levels in
biological samples, hepatic HT S50 of European chub and
brown trout and gills and the intestine of European chub
were measured by both parameter settings with the aim to
Table 1
The instrumental measuring
conditions set on 797
procedure by
Raspor et al.
Applied
in our study
–
–
–1
4 T et al.: iomonitoring
Croat. Chem. Acta , 91()
compare the obtained MT concentrations and evaluate the
application of Brdička reaction with the novel operating
parameters. As the method of differential pulse voltamme-
try (DPV) is generally very sensitive and the linearity range
is narrow, optimal conditions need to be used[12] and meas-
ured values in the unknown samples have to be in the range
of the determined calibration curves, preferably around the
central part of the linear calibration where the confidence
intervals are the narrowest. If measured sample concentra-
tions are not in the calibration range, then sample volume
or concentration have to be adjusted by addition of appro-
priate sample volume to the electrolyte solution, or by ap-
propriate sample dilution, respectively.
Since the preparation steps of HT S50 fraction for MT
measurement involved 60 times dilution of the initial fish
tissue, there was no need to additionally dilute HT S50 of
fish tissues prior to the measurement by both DPV
parameter settings if sample aliquots of 20–40 µL were
added to the electrolyte solution of 10 mL. Measurements
of fish samples showed that there were no significant
differences in MT levels in fish tissues obtained by the two
different parameter settings (Table 3). Coefficient of
determination (R2) between two measurements of
different scan rates for all analysed fish tissues was 0.94,
pointing to high correlation between them. The variability
in MT concentrations was 0.3–22 % when the
concentrations of the same fish tissues measured by two
parameter settings were compared.
Application and the Sensitivity of the
Modified Brdička Reaction
Modified Brdička reaction is a commonly used
electrochemical method for MT determination in different
biological samples.[4,21–23] Development of electrochemical
-
et al.–1
line
was – × 10
–
-
new
–1
straight line was –
–
T et al.: iomonitoring 5
Croat. Chem. Acta , 91()
methods have improved the quantification of analytes with
loweri ng the detectio n range to as low as 10–7 do 10–10 M.[4,23]
In our research, method was confirmed as sensitive, fast
and reliable technique for quantification of MTs in tissues
of freshwater fishes. The detection limit for MT was
evaluated as 3 nM. The change in the duration of one step
potential and scan rate have not influenced the limit of
detection, accuracy and reliability of the measurement
results, but contributed to faster measurements and
consequently resulted in time-saving modification. MT
levels in biological samples were highly comparable when
using both parameter settings - modified Brdička reaction
by Raspor et al.[13] (scan rate 0.0052 V s–1) and the new one
from this research (scan rate 0.013 V s–1).
Although there are many methods used for MT
quantification[4,5] one of the main advantages of the DPV
and Brdička procedure is that they require a small amount
of the samples to conduct the assay. This is especially useful
when small animals or small tissues (e.g. crustaceans,
bivalves, fish tissues) are applied as bioindicators, what is
often the case in the biomonitoring studies in aquatic
ecosystems. On the other hand, one of the main
disadvantages of the previously used procedure was that
the method was time-consuming, but presented research
showed that this problem can be overcome by decreasing
the duration of one step potential from 0.5 s to 0.2 s. As a
result of this change, the scan rate is faster and allows the
application of Brdička reaction in MT measurement in
biomonitoring studies, in which many samples of
bioindicator organisms have to be analysed.
CONCLUSIONS
Improvement of the modified Brdička reaction, which is
commonly used electrochemical method for the MT
Table
Squalius
cephalus
(
Salmo trutta
procedure by Raspor
et al–1)
rate: 0
–1
Fish tissues
–1
–1
–1
Squalius cephalus
1
Gills1
Salmo trutta
1
Table
–1 –1
) based
-
–1
–1
–1
–1
-
Average
– –
6 T et al.: iomonitoring
Croat. Chem. Acta , 91()
measurement, included the change in two measurement
parameters, duration of one step potential was lowered
from 0.5 s to 0.2 s, and scan rate was consequently
increased from 0.0052 V s–1 to 0.013 V s–1. Both applied
parameter settings meet the acceptance criteria set for
accuracy and precision, i.e. accuracy was –0.12 % and
–2.00 %, RSD was 1.89 % and 1.93 %, while recovery was
99.8 % and 98.0 %. Coefficient of determination (R
2)
between two parameter settings applied for the MT
measurement in different fish tissues was 0.94. Therefore,
presented changes in the duration of one step potential
and scan rate did not influence the accuracy and reliability
of the results, but had a positive impact on the duration of
reaction making the measurement much faster. This
improvement will especially be useful and applicable in
different biomonitoring studies which require the analyses
of numerous biological samples and a high sample
throughput, meaning the measurement of more samples in
a shorter time.
This work was supported by the Croatian
-09-
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