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Present study aims to improve the modified Brdička procedure of metallothionein (MT) quantification in a way to reduce the analysis duration. Therefore, scan rate of the voltammetric sweep was increased from 0.0052 V s –1 to 0.013 V s –1 by lowering the duration of one step potential from 0.5 s to 0.2 s, which resulted in 2.5 times faster voltammetric sweep. Research confirmed high accuracy and reliability of MT measurements, in both standard sample and samples of heat-treated cytosols of liver, gills and the intestine of European chub and liver of brown trout. The method was confirmed as fast and reliable electrochemical technique for quantification of MTs and this time-saving improvement is especially useful and applicable in different biomonitoring studies which require the analyses of numerous biological samples and high sample throughput.
This work is licensed under a 
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: 
  
Abstract: alysis
 –1 –1 
 
measurements, in both standard sample and sa-  
                 -saving
improvement is especially use
Keywords: ring
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
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
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.
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
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: (cMTaveragecMTstd) / 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.
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 solutionMT = 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
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.[ 1620] 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.
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 2040 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 0.322 % 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.[4,2123] Development of electrochemical
et al.–1
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 1010 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.
Improvement of the modified Brdička reaction, which is
commonly used electrochemical method for the MT
Table 
     
     
  
        
Salmo trutta     
procedure by Raspor
et al–1)
   
rate: 0
Fish tissues
Squalius cephalus 
1  
  
  
  
  
Gills1  
  
  
  
  
  
  
  
  
  
Salmo trutta 
1  
  
  
  
Table 
      
  
–1   –1
) based
       
 
 
 
 
 
 
 
 
 
 
 
 
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 s1 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
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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... The concentration of metallothionein (MT) proteins was determined in plasma. The quantification of MT concentration in the heat-treated samples was performed by differential pulse voltammetry following the methodology of the modified Brdička procedure (Raspor et al., 2001;Mijošek et al., 2018). All samples and MT standards were heat-treated and analysed in two technical replicas and a detailed description can be found in SI. ...
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The goal of the present study was to investigate differences in biomarker responses related to metal(loid)s in white stork Ciconia ciconia) nestling's blood from continental Croatia. To achieve this, a battery of biomarkers that can be affected by environmental pollutants, including metal(loid)s, was assessed (esterase activity, fluorescence-based oxidative stress biomarkers, metallothionein levels, glutathione-dependent enzyme activity). The research was conducted during the white stork breeding season in diverse areas (a landfill, industrial and agricultural sites, and an unpolluted area). White storks' nestlings near the landfill exhibited reduced carboxylesterase (CES) activity, elevated glutathione (GSH) concentration, as well as high Pb content in the blood. Increased As and Hg concentrations in blood were attributable to environmental contamination in agricultural area and an assumed unpolluted area, respectively. Furthermore, agricultural practices appeared to affect CES activity, as well as elevate Se levels. In addition to the successful implementation of biomarkers, present research showed that agricultural areas and a landfill are areas with increased metal(loid) levels possibly causing adverse effects on the white storks. This first-time heavy metal and metalloid analyses in the white stork nestlings from Croatia point to the necessary monitoring and future assessments of pollution impact to prevent irreversible adverse effects.
... Electrolyte solution consisted of 2 M NH 4 Cl/NH 4 OH and 1.2 × 10 − 3 M Co(NH 3 ) 6 Cl 3 (v/v 1:1), pH = 9.5, and was thermostated to 20 • C and purged with the pure nitrogen. Applied measurement parameters were adapted from Mijošek et al. (2018). Straight calibration line, constructed with the commercially available standard rabbit liver MT-2 (Enzo, USA), dissolved in 0.25 M NaCl, was used for the calculation of MT concentrations which were presented as mg MT g − 1 of wet tissue (w.w.). ...
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The application of invasive fish Prussian carp (Carassius gibelio Bloch, 1782) as bioindicator organism, using intestine as bioindicator tissue of anthropogenic influence in the lowland Ilova River was estimated. Intestinal tissue enables the investigation of dietborne metal uptake, so the first record on intestinal metal levels in Prussian carp was presented, as total and cytosolic fraction, which indicates the proportions of potentially toxic and bioavailable metals. Pollution impact was also estimated by analyses of biomarkers of oxidative stress (malondialdehyde), antioxidative capacity (catalase and glutathione) and of metal exposure (metallothioneins). All analyzed parameters were compared in the intestine of fish from the reference site and contaminated site impacted by technological and municipal wastewaters in two seasons. Both total and cytosolic As, Ca, Cd, Cs, Cu, Mg, Na and Rb levels were significantly higher at contaminated than the reference site in at least one season, whereas Mn and V had higher concentrations at the reference site. Despite differences in concentrations, average proportions of total metal levels in cytosolic fraction were comparable at two sites, i.e. over 70% for Na, K, Rb, Se, Cd, Cs, As and Mo, indicating their high possibility of binding to important biomolecules. In addition, higher levels of malondialdehyde in both seasons and enhanced catalase activity in spring, indicated disturbed environmental conditions near the contaminated site and need of continuous monitoring of this region. Finally, our research represents successful application of widely distributed invasive species in ecotoxicological studies, whereas intestine was shown as a suitable bioindicator tissue, clearly reflecting dietary metal uptake.
... Measurements were done in duplicate (A and B subsample) in 10 mL of an electrolyte solution consisting of 5 mL of 2 M NH 4 Cl/NH 4 OH and 5 mL of 1.2 × 10 −3 M Co (NH 3 )6Cl 3 , pH = 9.5 which was thermostated to 20 °C and purged with the pure nitrogen. The applied measurement parameters for DPV were the following: potential scan from −0.9 V to −1.65 V; scan rate 0.013 Vs −1 ; voltage pulse amplitude 0.02502 V; duration of the pulse application 0.057 s and a step time 0.2 s ( Mijošek et al., 2018). MT concentrations were derived from the straight calibration line, constructed with the commercially available standard rabbit liver MT-2 (Enzo, USA) dissolved in 0.25 M NaCl. ...
Application of metallothionein (MT) as an early warning sign of metal exposure in aquatic organisms is common in biomonitoring, but there is a huge variability in MT concentrations among different studies. Present research aims to assess MT responses in freshwater fish brown trout (Salmo trutta Linnaeus, 1758) and gammarids (Gammarus balcanicus Schäferna, 1922 and Echinogammarus acarinatus Karaman, 1931) as indicators of metal exposure within the freshwater karst environment (Krka River, Croatia). Sampling was performed upstream (reference site) and downstream (anthropogenically impacted site) of the wastewater discharges in autumn and spring seasons. Brown trout intestine was applied as a bioindicator tissue due to its role in dietborne metal uptake while gammarids were chosen as fish food and potential metal uptake source. Moreover, there is a lack of data on intestinal MT levels, so our results on MT and metal/metalloid concentrations, measured as total and metabolically available cytosolic levels, represent the first data of this kind for the selected indicator species. The results indicated that the ecotoxicological impact of technological and municipal wastewaters on the biota of the karst Krka River was moderate, although higher metal levels at the affected site were evident in both, fish and gammarids. The modified Brdička reaction applied in this study was confirmed as reliable electrochemical technique for MT quantification in both vertebrates and invertebrates, and it indicated higher MT levels in gammarids (1.9-4.1 mg g −1 w.w) than in fish intestine (0.5-2.8 mg g −1 w.w). Due to the lack of the data on MT concentrations in S. trutta and gammarid species G. balcanicus and E. acarinatus, presented results can serve as a preliminary data to establish MT background levels in intestine of wild freshwater fish and gammarids. Obtained MT levels showed species-, tissue-and method-specific differences, so comparison between MT levels should always involve the same species, tissue and measurement method.
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Metallothioneins (MTs) are a group of low molecular mass, cysteine-rich proteins with a variety of functions including involvement in metal homeostasis, free radical scavenging, protection against heavy metal damage, and meta-bolic regulation via Zn donation. The overexpression of MTs in proliferating cells is turning the attention to the study of MTs as novel promising marker of tumor diseases. Besides, levels of MTs in invertebrates and aquatic vertebrates well correlate with heavy metal pollution of an environment and, thus, serve as bio-environmental marker. It is not surprising that these proteins are of great interest not only for biochemists, molecular biologists clinical chemists, but also for envi-ronmental chemists and ecologists. Detection and quantification of MTs, which is needed in all above mentioned areas, is challenging due to the high cysteine content and relatively low molecular mass. This review is giving an overview of the analytical methods used for determi-nation of MTs comprising a broad range of immunochemical, electrochemical as well as spectrometric methods, which have been optimized and even hyphenated with different separation methods to detect MTs. More attention is paid to the structural analysis of these proteins as well as the employment of analytical instruments for environmental, biochemical and clinical purposes.
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Among wide spectrum of biomolecules induced by various stress factors low molecular mass protein called metallothionein (MT) is suitable for assessment of the heavy metal environmental pollution. The aim of this work was to determine the metallothionein and total thiols content in larvae of freshwater midges (Chironomus riparius) sampled from laboratory exposure to cadmium(II) ions and from field studies using differential pulse voltammetry Brdicka reaction. Unique electrochemical instrument, stationary electrochemical analyser Autolab coupled with autosampler, was utilized for the analysis of the samples. The detection limit for MT was evaluated as 5 nM. The larvae exposed to two doses (50 ng/g or 50 μg/g) of cadmium(II) ions for fifteen days under laboratory controlled conditions were at the end of the exposure killed, homogenized and analysed. MT content in control samples was 1.2 μM, in larvae exposed to 50 ng Cd/g it was 2.0 μM and in larvae exposed to 50 μg Cd/g 2.9 μM. Moreover at field study chironomid larvae as well as sediment samples have been collected from eight field sites with different levels of pollution by heavy. The metals content (chromium, nickel, copper, zinc, arsenic, molybdenum, cadmium, tin and lead) in the sediment and or MT content in the chironomid larvae were determined by inductively coupled plasma mass spectrometry or Brdicka reaction, respectively.
The Brdička reaction has been widely used for protein analysis, even though its mechanism was not completely elucidated. It is usually performed in a solution of ammonia buffer and Co(NH3)6Cl3 in differential pulse (DP) mode at the mercury electrode. A new interpretation of the existing results is presented leading to the complete elucidation of the mechanism. The signal with Ep=−1.35 V versus the reference electrode corresponds to the reduction of the complex RS2Co, while the only signal which corresponds to the reduction of the hydrogen at the mercury electrode is the most negative one at Ep=−1.48 V. Based on the mechanism elucidated, it clearly follows that the Brdička reaction is a complex one. Therefore, the crucial physico-chemical parameters like the temperature, pH, c(NH4+), c(Co(NH3)6Cl3), c(protein) should be optimized and, in an analytical protocol, kept strictly in order to obtain intercomparable results on metallothionein analysis.
Quantitative analysis of metallothioneins (MTs) by the modified Brdička procedure is based on the evaluation of the catalytic hydrogen signal (Cat). The effect of the basic physico–chemical parameters, the temperature and the depolarizer concentration (Co(NH3)6Cl3), on the Cat signal height and the respective calibration straight lines was studied by means of the differential pulse (DP) mode at the hanging mercury drop electrode (HMDE). The temperature was varied from 7 to 18°C, and the depolarizer concentration from 1.2×10−4 to 1.0×10−3 M Co(NH3)6Cl3. The commercially available rabbit liver MT was used as the calibrant. The parameters of the calibration straight lines, the standard errors of the intercept sa and the slope sb were evaluated by means of the least-square method. The hypothesis of a significant difference of the estimated value of the intercept on y-axis compared to the zero value was statistically tested. For those conditions for which this hypothesis was rejected the measuring conditions of the catalytic hydrogen signal are selected and they refer to a buffer 1M (NH4Cl+NH4OH), pH 9.5, (7.0±0.5)°C and the depolarizer concentration (0.8 or 1.0)×10−3 M Co(NH3)6Cl3.
A six-to nine-fold increase in detection limits of the Brdic̈ka polarographic assay for metallothioneins has been achieved through the employment of a commercially available static mercury drop electrode (SMDE) and reduced temperatures. At 16°C detectable concentrations of metallothionein (MT-1) from the crab Scylla serrata were in the range of 5 to 10 μg litre-1. Operating conditions and electrode preparation are discussed. For a number of thiolic molecules a linear relationship between polarographic response and either the number of cysteinyl residues or the molecular weight was noted. In the latter case on enhancement of response by sulfur-metal bonds is suggested. The utility of this assay permits a rapid quantitative assay for these proteins at basal levels in marine invertebrates and other organisms.
As a result of mining, forestry, waste disposal and fuel combustion, our environment is becoming increasingly contaminated with heavy metals. The aquatic environment receives waste products from such activities and may be the final depository for these anthropogenically remobilized heavy metals. In order to understand the impact of heavy metals on aquatic biota it is important to characterize the mechanisms available for aquatic life to transport, immobilize and excrete heavy metals.
The assessment of general condition of fish in the moderately contaminated aquatic environment was performed on the European chub (Squalius cephalus) caught in September 2009 in the Sutla River in Croatia. Although increases of the contaminants in this river (trace and macro elements, bacteria), as well as physico-chemical changes (decreased oxygen saturation, increased conductivity), were still within the environmentally acceptable limits, their concurrent presence in the river water possibly could have induced stress in aquatic organisms. Several biometric parameters, metallothionein (MT), and total cytosolic protein concentrations in chub liver and gills were determined as indicators of chub condition. Microbiological and parasitological analyses were performed with the aim to evaluate chub predisposition for bacterial bioconcentration and parasitic infections. At upstream river sections with decreased oxygen saturation (∼50 %), decreased Fulton condition indices were observed (FCI: 0.94 g cm(-3)), whereas gonadosomatic (GSI: 2.4 %), hepatosomatic (HSI: 1.31 %), and gill indices (1.3 %) were increased compared to oxygen rich downstream river sections (dissolved oxygen ∼90 %; FCI: 1.02 g cm(-3); GSI: 0.6 %; HIS: ∼1.08 %; gill index: 1.0 %). Slight increase of MT concentrations in both organs at upstream (gills: 1.67 mg g(-1); liver: 1.63 mg g(-1)) compared to downstream sites (gills: 1.56 mg g(-1); liver: 1.23 mg g(-1)), could not be explained by induction caused by increased metal levels in the river water, but presumably by physiological changes caused by general stress due to low oxygen saturation. In addition, at the sampling site characterized by inorganic and fecal contamination, increased incidence of bacterial bioconcentration in internal organs (liver, spleen, kidney) was observed, as well as decrease of intestinal parasitic infections, which is a common finding for metal-contaminated waters. Based on our results, it could be concluded that even moderate contamination of river water by multiple contaminants could result in unfavourable living conditions and cause detectable stress for aquatic organisms.
The subcellular distribution of cadmium, copper and zinc in the liver, and the binding of these heavy metals to metallothionein (MT) were studied in a wild population of perch (Perca fluviatilis). The fish were caught in two areas of the cadmium-contaminated river Emån in the southeast of Sweden. The livers were analyzed for zinc, copper and cadmium and the subcellular distribution of the heavy metals in mitochondrial, microsomal and cytosolic fractions was determined. The cytosols were chromatographed on Sephadex G-75 columns to determine the partition of heavy metals between high molecular weight and MT fractions. Metallothionein was determined, in tissue extracts, by differential pulse polarography. The elevated hepatic cadmium levels found in perch from the contaminated region of the river did not significantly alter the distribution of zinc and copper in the liver. With increasing amounts of cadmium present in the liver cytosol, the distribution of cadmium was altered. The increased cadmium content correlated with an increased MT level in the liver (r=0.84). The relationship between cadmium and MT is described by the formula [MT] = 7.2 + 0.53 [Cd]. Chromatography of the liver revealed that all of the applied cadmium was bound to MT. The variations in zinc and copper showed low correlation to the MT content of the liver (r=0.51 and r=−0.15, respectively).
Metallothioneins (MTs) are a family of ubiquitous, biologically interesting proteins that have been isolated and studied in a wide variety of organisms, including prokaryotes, plants, invertebrates and vertebrates. Due to the property of MTs being metal-inducible and their high affinity to metal ions, homeostasis of heavy-metal levels is probably their most important biological function.MTs are also involved in other important biochemical pathways, including scavenging of reactive oxygen species, activation of transcription factors and participation in carcinogenesis. Detection and quantification of MTs are not simple due to the unique primary structure and their relatively low molecular mass. Analytical methods are based on: a) detection of bound metal ion; b) detection of free –SH groups; c) protein mobility in electrical field; and, d) interaction with different types of sorbent.This review highlights techniques used for detection and determination of MTs with discussion of the advantages and the disadvantages of particular approaches.