Using of Electrochemical Methods for Studying of Metallothionein Content in the Human Blood Serum of a Patient Poisoned by Lead and Treated by Platinum

Metallothioneins belong to the group of intracellular, high molecular and cysteine-rich proteins whose content increase with increasing concentration of a heavy metal. Here we applied the adsorptive transfer stripping differential pulse voltammetry Brdicka reaction for the determination of metallothionein in human blood serum of patient poisoned by lead and/or treated by platinum. The increased metallothionein concentrations in both cases were observed.


INTRODUCTION
Metallothionein (MT) belongs to the group of intracellular, high molecular and cysteine-rich proteins with molecular weight from 6 to 10 kDa (ref. 1 ).The MT was discovered in 1957, when Margoshes and Valee isolated it from horse kidney 2 .It is known that aromatic amino acids are not present in the MT molecule and twenty cysteines usually occur in its primary sequence at these repetitions: Cys-X-Cys, Cys-Cys-X-Cys-Cys, Cys-X-Cys-Cys, where X represents other aminoacid than cysteine 1,3 .MTs consist of two binding domains (α, β) that are assembled from cysteine clusters.Cysteine sulfhydryl groups participate in covalent bindings with heavy metals.The N-terminal part of the protein is marked as α-domain, which has three binding places for divalent ions.β-Domain (C-terminal part) has the ability to bind four divalent ions of heavy metals.Moreover a group of human metallothioneins such as MT1, MT2, MT3 and MT4 has been described (Table 1).
Number of analytical techniques including electrochemistry [4][5][6][7][8] is used for the determination of MT.Many authors have been giving attention to electrochemical determination and study of MT for more than 20 years [9][10][11][12] .In addition, more than 70 years ago Brdicka discovered the catalytic evolution of hydrogen in the presence of cobalt(III) solution and of proteins [13][14][15][16] .This reaction has been intensively using for electrochemical determination of proteins and has been modified by number of authors [17][18][19] .
The aim of this work was to use adsorptive transfer stripping (AdTS) differential pulse voltammetry (DPV) Brdicka reaction for studying of metallothionein content in the human blood serum of a patient poisoned by lead and treated by platinum.

Chemicals
Sodium chloride and other used chemicals were purchased from Sigma Aldrich.The stock standard solutions of MT at 10 µg/ml was prepared by ACS water (Sigma-Aldrich, USA) and stored in the dark at -20 °C.Working standard solutions were prepared daily by dilution of the stock solutions.The pH value was measured using WTW inoLab Level 3 with terminal Level 3 (Weilheim, Germany), controlled by personal computer program (MultiLab Pilot; Weilheim, Germany).The pH-electrode (SenTix-H, pH 0-14/3M KCl) was regularly calibrated by set of WTW buffers (Weilheim, Germany).

Human blood serum samples
Human blood serum samples were obtained from the Department of Clinical Biochemistry and Pathobiochemistry, 2 nd Faculty of Medicine Charles University, Czech Republic.The samples were prepared by heat treatment and solvent precipitation.Briefly, the samples were kept at 99 °C in a thermomixer (Eppendorf 5430, USA) for 15 min with occasional stirring, and then cooled to 4 o C. The denatured homogenates were centrifuged at 4 o C, 15 000 g for 30 min.(Eppendorf 5402, USA).Heat treatment and solvent precipitation effectively denature and remove high molecular weight proteins out from samples 19,20 .Determination of MT in the human blood serum samples was performed by optimised AdTS DPV Brdicka reaction.Analysed sample volume was 5 µl.

Electrochemical measurements
Electrochemical measurements were performed with AUTOLAB Analyser (EcoChemie, Netherlands) connected to VA-Stand 663 (Metrohm, Switzerland), using a standard cell with three electrodes.The working electrode was a hanging mercury drop electrode (HMDE) with a drop area of 0.4 mm 2 .The reference electrode was an Ag/ AgCl/3M KCl electrode and the auxiliary electrode was a graphite electrode.The supporting electrolyte was prepared by mixing buffer components except sodium chloride.For smoothing and baseline correction the software GPES 4.4 supplied by EcoChemie was employed.

AdTS DPV Brdicka reaction of MT
The Brdicka supporting electrolyte containing 1 mM Co(NH 3 ) 6 Cl 3 and 1 M ammonia buffer (NH 3 (aq) + NH 4 Cl, pH = 9.6) was used; surface-active agent was not added.AdTS DPV Brdicka reaction parameters were as follows: an initial potential of -0.35 V, an end potential -1.6 V, a modulation time 0.057 s, a time interval 0.2 s, a step potential of 1.05 mV/s, a modulation amplitude of 250 mV, E ads = 0 V. Temperature of supporting electrolyte was tested 20 .

RESULTS AND DISCUSSION
Brdicka discovered protein catalysed hydrogen evolution signals more than 70 years ago 21 .In addition Brdicka found out that the hydrogen evolution could be used for diagnostic of tumour diseases 22,23 .On the other hand this technique has been replaced by new analytical techniques such electrophoresis, chromatography coupled with different kind of detectors in clinical medicine.Therefore we were interested in the issue if Brdicka reaction could be used in clinical lab nowadays.Recently new approaches such as adsorptive transfer stripping technique or modification of a surface of working electrode by a high-and/or low-molecular compound (Fig. 1) used for improvement of selectivity of Brdicka reaction has been described 24,25,26 .If the authors used the adsorptive transfer stripping technique in combination with Brdicka reaction, the sensitive determination of proteins was reached 27,20 .
Here we wanted to analyse the human blood serum samples of a patient poisoned by lead.To be specific, a 11-year-old girl was referred to the hospital by her general practitioner because of abdominal pain, vomiting, dark colour of tongue, low intake of fluid and food, and abnormal laboratory results (bilirubin 55 µmol/l, AST 2.94 µkat/l, ALT 3.46 µkat/l, haemoglobin 86 g/l).Blood film, which  exhibited basophilic stippling, is prompting for investigation of lead poisoning.The source of 6 months lead exposure was identified as tea from a ceramic tea pot with insufficient glassing (lead concentration was 45332 µg/l in tea after 30 minutes).It is common knowledge that heavy metals induce a synthesis of MT.Therefore we assumed that concentration of MT increased with rising amounts of lead in blood serum of the poisoned patient.The AdTS DPV signals i) of reduction of Co(II) contained in supporting electrolyte (dot-and-dash line); ii) of control (dashed line) and iii) of human blood serum of poisoned patient -first day of hospitalisation (continuous line) are shown in Fig. 2A.We observed three MT signals -RS 2 Co (complex of MT with Co(II)) and two catalytic signals (Cat1 and Cat2).We estimated that amount of MT in a blood serum of a healthy patient varies between 1 and 10 µM (n = 5).It clearly follows from the figure that Cat2 signal increased according to lead poisoning.Finally, it was found out that amount of MT in the human blood serum of the lead poisoned patient was more than 200 µM.
Using of electrochemical methods for studying of metallothionein content in the human blood serum of a patient poisoned by lead and treated by platinum Moreover MT concentration increase during treating of tumour diseases by platinum based cytostatics 28 .Therefore we determined the MT amounts in human blood serum of a patient, which has been treated by platinum complexes, by AdTS DPV Brdicka reaction.The increase in Cat 2 signal height of the treated blood serum corresponds to MT concentration of 150 µM (Fig. 2B).That is why it is possible to assume that content of metallothionein was increased due to treatment by platinum based cytostatic.Moreover we observed a signal at potential of -0.7 V, which probably corresponds to complex between MT and platinum cytostatic.The experimental data indicate that it would be suitable to monitor the content of MT at patients treated by platinum based cytostatics for better recognising of platinum cytostatic resistance 29 .