Determination of Metal Ions in the Plasma of Children with Tumour Diseases by Differential Pulse Voltammetry

In . J. Elec ochem. Sci., 9 (2014) 4675 - 4691 In e na ional Jou nal o ELECTROCHEMICAL SCIENCE www.elec ochemsci.o g De e mina ion o Me al Ions in he Plasma o Child en wi h Tumou Diseases by Di e en ial Pulse Vol amme y Rena a Kenso a1,2, Da id Hynek1,2,3, Jind ich Kynicky3,4, Ma ie Konecna1,2,3, Tomas Eckschlage 5, Voj ech Adam1,2,3, Ja omi Hubalek2,3, Rene Kizek1,2,3* 1 Depa men o Chemis y and Biochemis y, Facul y o Ag onomy, Mendel Uni e si y in B no, Zemedelska 1, CZ-613 00 B no, Czech Republic, Eu opean Union 2 Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, Technicka 3058/10, CZ- 616 00 B no, Czech Republic, Eu opean Union 3 Lead and Cadmium Ini ia i es, Uni ed Na ion En i onmen P og am, Facul y o Ag onomy, Mendel Uni e si y in B no, Zemedelska 1, CZ-613 00 B no, Czech Republic, Eu opean Union 4 Ka el Englis College, Sujano o nam. 356/1, CZ-602 00, B no, Czech Republic, Eu opean Union 5 Depa men o Paedia ic Haema ology and Oncology, 2nd Facul y o Medicine, Cha les Uni e si y, and Uni e si y Hospi al Mo ol, V U alu 84, CZ-150 06 P ague 5, Czech Republic, Eu opean Union *E-mail: [email p o ec ed] Recei ed: 21 Ma ch 2014 / Accep ed: 5 Ap il 2014 / Published: 19 May 2014 The aim o his s udy was o in es iga e he in e ac ion be ween lead ions and e hylenediamine e aace ic acid (EDTA) in plasma. EDTA is widely used in medicine as an icoagulan agen . Unde s anding o he in luence o EDTA on he elec ochemical lead ions de e mina ion is e y sui able o he de e mina ion o lead ions a pa ien s wi h inc eased me als le els in body luids. Fu he , he changes o me al ions le els (Zn, Cd, Pb and Cu) we e moni o ed in he blood plasma o child pa ien s ea ed o a ious oncological diseases. Elec ochemical me hod di e en ial pulse ol amme y wi h ully au oma ed sys em and a omic abso p ion spec ome y was used o de e mina ion o he me al ions. I was ound an inc eased amoun o me al ions in he blood plasma o pa ien s su e ing om cance disease in compa ison wi h physiological alues in heal hy people. In hese pa ien s he e was also ound ha hey ha e highe le els o me allo hionein and he a io o GSH/GSSG was educed. These esul s sugges ha umou diseases cause impo an changes in he le el o ions. Keywo ds: Me als; Elec ochemical Analysis; Plasma; EDTA; In e ac ion; Oncological Diseases 1. INTRODUCTION Human exposu e o me als is common due o wide p esence in indus y and long- e m en i onmen al pe sis ence. Among he gene al popula ion, exposu e o a numbe o me als is In . J. Elec ochem. Sci., Vol. 9, 2014 4676 widesp ead bu gene ally a subs an ially lowe le els han ha e been ound in indus y. Accumula ion o me al ions in a y issues and ci cula o y sys em, nega i e e ec s on cen al ne ous sys em and unc ioning o in e nal o gans as well as ac ing as igge s o se e al se ious diseases including umou ones can be lis ed as ad e se e ec s o me al ions on humans [1,2]. Me als sha e ce ain physical and chemical ea u es and i is easonable o specula e ha common mechanisms o ca cinogenici y may ake place. Speci ic ca cinogenic pa hways, howe e , a e de e mined by nume ous ac o s including me al ype, specia ion, solubili y, possible me al-me al in e ac ions, and o he s [3,4]. The ca cinogenici y o a senic(III), ch omium(VI), and nickel(II) has been con i med in humans [3]. Some expe imen al and epidemiologic s udies sugges ha lead may be a human ca cinogen, bu he e idence is inconclusi e so a . Al hough epidemiologic da a a e less ex ensi e o be yllium(II) and cadmium(II), he indings in humans o excess cance isk a e suppo ed by he clea demons a ion o ca cinogenici y in expe imen al s udies [5]. O he me als, including an imony and cobal , may be ca cinogens in human, bu he expe imen al and epidemiologic da a a e limi ed ye [3]. Hea y me al adso p ions om he diges ion sys em and haemoglobin sensi i i y o hese me als a e much highe in child en compa ed o adul s [6-9]. The heal h isk is especially high o child en, because o hei ole ance o poisons is lowe [10]. Fu he , ch onic e ec s o me als ha migh no be immedia ely appa en ep esen an impo an issue ha also needs o be aken in o accoun . Thanks o he nega i e cha ac e is ics e e ed o me als, i is necessa y o moni o hei amoun in he body [11- 14]. In ha case, i is o en necessa y o analyse complex biological ma ices such as blood, se um, issue and u ine. A a ie y o me hods has been epo ed in he li e a u e, including induc i ely coupled plasma – mass spec ome y (ICP-MS) and o he spec oscopic and elec ochemical me hod [15-17]. I is clea ha he moni o ing o me als in biological samples is gi en g ea a en ion [12,18,19]. Blood as a biological ma e ial is collec ed mos equen ly o labo a o y es ing. The way o sampling is gi en by me hodology o examina ion in he labo a o y and i s echnical equipmen . Fo haema ological blood es ing, he examined samples a e whole blood, plasma o se um. Du ing an analysis o plasma he e is necessa y o add he an icoagulan solu ion o he blood sample. The an icoagulan composi ions ake he o m o liquid (mixed in a ce ain a io wi h pa ien blood in collec ion ube) o a c ys alline o m ( he c ys alline e apo a ion esidue on he walls o collec ion ube). The mos common an icoagulan agen s include hepa in, sodium ci a e 3.8% and K2 o K3 EDTA (di- o i-po assium sal o e hylenediamine e aace ic acid) [20-26]. In he sampling ubes (VACUETTE®) o haema ology, he EDTA d y addi i e is applied o he inne wall. Al e na i ely, hese ubes can con ain an 8% liquid EDTA solu ion (0.3 µM). The EDTA binds calcium ions and hus blocked he coagula ion cascade [26]. Besides EDTA an icoagulan e ec , his subs ance has he abili y o bind o he me al ions. The e o e he in luence o EDTA o he me al ions de e mina ion was s udied. Elec ochemical echniques we e used o me al ions quan i ica ion because hey belong o he bes me hods o me al ions de ec ion. These me hods ha e ad an ages associa ed wi h high sensi i i y, low de ec ion olumes, compac ins umen a ion and low cos [27-31]. Mo eo e , he op imized me hods we e applied o analyse blood samples ob ained om pa ien s wi h a umou disease. In . J. Elec ochem. Sci., Vol. 9, 2014 4677 2. EXPERIMENTAL PART 2.1. Chemicals All chemicals used in his s udy we e pu chased om Sigma Ald ich (S . Louis, MA, USA) in ACS pu i y unless no ed o he wise. HPLC-g ade me hanol (>99.9%; / ) was ob ained om Me ck KGaA (Da ms ad , Ge many). Pipe ing was pe o med by pipe es om Eppendo (Hambu g, Ge many). Wo king s anda d solu ions we e p epa ed daily by dilu ing he s ock solu ions. S ock solu ions o me als (1 mg/ml) was p epa ed by dissol ing app op ia e amoun o zinc(II) ni a e, cadmium(II) ni a e, lead(II) ni a e and coppe (II) ni a e in ACS wa e and dilu ed o 50 ml olume ic lask. Ace a e bu e o pH 5 was p epa ed wi h 0.2 M ace ic acid and 0.2 M sodium ace a e and dilu ed wi h wa e and used as a suppo ing elec oly e. High pu i y deionised wa e (Milli-Q Millipo e 18.2 MΩ/cm, Bed o d, MA, USA) was used h oughou he s udy. The e was also used human blood plasma o sample p epa a ion. 2.2. P epa a ion o deionised wa e and pH measu emen The deionised wa e was p epa ed using e e se osmosis equipmen Aqual 25 (B no, Czech Republic). The deionised wa e was u he pu i ied by using appa a us MiliQ Di ec QUV equipped wi h he UV lamp. The esis ance was 18 MΩ. The pH was measu ed using pH me e WTW inoLab (Weilheim, Ge many). Deionized wa e was used o insing, washing, and bu e p epa a ion. 2.3. P epa a ion o samples Blood plasma samples we e ob ained om 10 child in pa ien s a Depa men o Paedia ic Haema ology and Oncology o Facul y Hospi al Mo ol wi h newly diagnosed solid umou s (non- Hodgkin lymphoma (n=2), Ewing sa coma, hepa oblas oma, es icula ge m cell umou , emb yonal habdomyosa coma, neu oblas oma (n = 2), medulloblas oma, neph oblas oma; a e age age o 12.2 yea s). The samples we e s o ed in -80 °C un il assayed. 2.3.1. Mic owa e diges ion o elec ochemical and spec ome ic de e mina ion o me al ions 10 µl o blood plasma was pipe ed in o diges ion ials. Ni ic acid (65 %, / ) and hyd ogen pe oxide (30 %, / ) we e used as he diges ion mix u e. The e was used 500 µl olume o his mix u e, while he olume a io be ween ni ic acid and hyd ogen pe oxide was always 7:3 (350 µl HNO3 and 150 µl H2O2). Samples we e diges ed by Mic owa e 3000 (An on Paa GmbH, Aus ia) using o o MG-65. The p og am begins and ends wi h he same en-minu e-long-s ep, beginning wi h he powe o 50 W and ending wi h he powe 0 W (cooling). Mic owa e powe was se o 100 W in he main pa o he p og ams (30 minu es) a empe a u e o 140 °C. A e he diges ion, he samples wi h diges ion mix u e we e pipe ed in o Eppendo ials and elec ochemical de e mina ion o zinc, lead, cadmium and coppe ollowed [27,32,33]. In . J. Elec ochem. Sci., Vol. 9, 2014 4678 2.3.2. Samples p epa a ion o me allo hionein elec ochemical de e mina ion Samples we e p epa ed by hea ea men by he help o he au oma ed pipe ing sys em epMo ion 5075 (Eppendo , Ge many), because he hea ea men e ec i ely dena u es and emo es he high molecula weigh p o eins om eal samples [34]. B ie ly, he samples we e kep a 4°C hen samples we e ans e ed o he 96 well pla es (Eppendo ) con aining 0.2 M phospha e bu e pH 7. This mix u e was kep a 99 °C o 15 min. The las s ep was cooling down o samples o 4 °C. The cooled samples we e hen cen i uged o 30 minu es a 15000 × g a 4 °C. The supe na an was collec ed and measu ed. 2.3.3. Samples p epa a ion o educed and oxidized glu a hione de e mina ion Se um was sepa a ed om whole blood by cen i uga ion a 4000 × g o 10 min (Model 5402, Eppendo , Ge many), and he samples we e s o ed a -80˚C un il assayed. When equi ed, he dena u ed samples we e cen i uged a 15000 × g a 4˚C o 30 min (Model 5402; Eppendo AG) and di ec ly analysed using an op imised high pe o mance liquid ch oma og aphy wi h elec ochemical de ec ion. P io o ch oma og aphic analysis, p ecipi a ion o p o eins wi h i luo oace ic acid (TFA) o a oid excessi e clogging o il e s and p ecolumns, which p o ec he sepa a ion column om con amina ions, was equi ed. The dena u ed sample (100 µl o plasma and 100 µl o 10% ( / ) TFA) was han cen i uged and he esul ing supe na an was di ec ly injec ed o he ch oma og aphic column. 2.4. A omic abso p ion spec ome y (AAS) Measu emen s we e ca ied ou on 240 FS AA Agilen Technologies lame a omic abso p ion spec ome e wi h deu e ium lamp backg ound co ec ion o 280Z Agilen Technologies a omic abso p ion spec ome e (Agilen , USA) wi h elec o he mal a omiza ion and Zeeman backg ound co ec ion. Zinc, cadmium, lead and coope we e measu ed on p ima y wa eleng hs: Zn 213.9 nm (spec al bandwid h 1.0 nm, lamp cu en 5 mA); Cd 228.8 nm (spec al bandwid h 0.5 nm, lamp cu en 4 mA); Pb 217.0 nm (spec al bandwid h 1.0 nm, lamp cu en 10 mA) and Cu 324.8 nm (spec al bandwid h 0.5 nm, lamp cu en 4 mA). Elemen s measu ed by elec o he mal AAS we e de e mined in he p esence o palladium chemical modi ie . The samples we e modi ied in acco dance wi h chap e 2.3.1. 2.5. De e mina ion o Zn, Cd, Pb and Cu in plasma o child en wi h malignan umou s De e mina ion o zinc, cadmium, lead and coope by di e en ial pulse ol amme y we e pe o med wi h 797 VA Compu ace ins umen connec ed o 813 Compac Au osample (Me ohm, Swi ze land), using a s anda d cell wi h h ee elec odes. The h ee elec ode sys em consis ed o a hanging me cu y d op elec ode (HMDE) wi h a d op a ea o 0.4 mm2 as he wo king elec ode, an Ag/AgCl/3 M KCl e e ence elec ode and a pla inum as he auxilia y elec ode. 797 VA Compu ace In . J. Elec ochem. Sci., Vol. 9, 2014 4679 so wa e by Me ohm CH was employed o da a p ocessing. The analysed samples we e deoxygena ed p io o measu emen s by pu ging wi h a gon (99.999%). Ace a e bu e (0.2 M CH3COONa + 0.2 M CH3COOH, pH 5) was used as a suppo ing elec oly e. The suppo ing elec oly e was eplaced a e an analysis. The pa ame e s o he measu emen we e as ollows: pu ging ime 90 s, deposi ion po en ial -1.15 V, accumula ion ime 240 s, equilib a ion ime 5 s, modula ion ime 0,057 s, in e al ime 0.04 s, ini ial po en ial o -1.3 V, end po en ial 0.2 V, s ep po en ial 0.005 V, modula ion ampli ude 0.025 V, olume o injec ed sample: 15 µl, olume o measu emen cell 2 ml (15 μl o sample and 1985 μl ace a e bu e ). Mo eo e , lead was de e mined in he p esence o EDTA by di e en ial pulse ol amme y (DPV) using he same ins umen and condi ions men ioned in he p e ious pa ag aph wi h he ollowing excep ions: ini ial po en ial o –0.6 V, end po en ial 0.2 V, deposi ion po en ial -0.5 V, accumula ion ime 300 s, deoxygena ing wi h a gon 90 s, olume o injec ed sample: 500 µl, olume o measu emen cell 2 ml (500 µl o sample and 1500 µl ace a e bu e pH = 5.0). 2.6. De e mina ion o me allo hionein in plasma o child en wi h malignan umou s Di e en ial pulse ol amme ic measu emen s we e pe o med wi h 747 VA S and ins umen connec ed o 693 VA P ocesso and 695 Au osample (Me ohm, Swi ze land), using a s anda d cell wi h h ee elec odes and cooled sample holde and measu emen cell o 4 °C (Julabo F25, JulaboDE). A hanging me cu y d op elec ode (HMDE) wi h a d op a ea o 0.4 mm2 was used as he wo king elec ode. An Ag/AgCl/3M KCl elec ode was he e e ence and pla inum elec ode was auxilia y. Fo da a p ocessing VA Da abase 2.2 by Me ohm CH was employed. The analysed samples we e deoxygena ed p io o measu emen s by pu ging wi h a gon (99.999 %) sa u a ed wi h wa e o 120 s. B dicka suppo ing elec oly e con aining 1mM Co(NH3)6Cl3 and 1M ammonia bu e (NH3(aq) + NH4Cl, pH = 9.6) was used. The suppo ing elec oly e was exchanged a e an analysis. The pa ame e s o he measu emen we e as ollows: ini ial po en ial o -0.7 V, end po en ial o -1.75 V, modula ion ime 0.057 s, ime in e al 0.2 s, s ep po en ial 2 mV, modula ion ampli ude -250 mV, Eads = 0 V, olume o injec ed sample: 10 µl, olume o measu emen cell 2 ml (10 μl o sample and 1990 ml o B dicka solu ion). 2.7. De e mina ion o GSH and GSSG in plasma o child en wi h malignan umou s The HPLC-ED sys em consis s o wo ch oma og aphic pumps (Model 582; ESA, Inc., Chelms o d, MA, USA; wo king ange 0.001-9.999 ml/min), a ch oma og aphic column wi h e e se phase Zo bax eclipse AAA C18 (Agilen Technologies, Inc., San a Cla a, CA, USA; 150×4.6 mm; 3.5- µm pa icles) and a wel e-channel CoulA ay elec ochemical de ec o (Model 5600A; ESA, Inc.). The de ec o consis s o h ee low analy ical chambe s (Model 6210; ESA, Inc.). A chambe con ains ou analy ical cells and one analy ical cell con ains wo e e en (hyd ogen-palladium), as well as wo coun e s and po ous g aphi e wo king elec odes. The ED is si ua ed in he he mos a ed con ol module. A 20 µl sample was injec ed using an au osample (Model 542; ESA, Inc.), which has In . J. Elec ochem. Sci., Vol. 9, 2014 4680 he mos a ed space o he column. The column was e mos a ed a 35˚C. Flow a e o mobile phase was 1 ml/min. The mobile phase consis ed o A: i luo oace ic acid (80 mM), and B: 100% me hanol ( / ). Subs ances we e elu ed wi h he ollowing linea inc easing g adien : 0-1 min (3% B) 1-2 min (10% B), 2-5 min (30% B), 5-6 min (98% B). De ec ion o he sepa a ed compounds was ca ied ou a he applied po en ial o 900 mV. Analysis ime was 20 minu es. 2.8. Ma hema ical ea men o da a and es ima ion o de ec ion limi s Da a we e p ocessed using MICROSOFT EXCEL (Mic oso , Redmond, WA, USA) and STATISTICA.CZ Ve sion 8.0 (S a -So CR, P ague, Czech Republic). The esul s a e exp essed as mean ± s anda d de ia ion (SD) unless o he wise no ed. The de ec ion limi s (3 signal/noise, S/N) we e calcula ed o Long and Wine o dne [35], whe eas N was exp essed as a s anda d de ia ion o noise de e mined in he signal domain unless o he wise s a ed. 3. RESULTS AND DISCUSSION 3.1. Op imiza ion o lead de e mina ion Among he di e en elec ochemical echniques ol amme ic and po en iome ic echniques a e he mos epo ed o hea y me als de ec ion [27,30,32,33,36-44]. In addi ion o con en ional hanging me cu y d op elec ode he mos commonly used elec ode ma e ial o he de ec ion o me als is ca bon. De e mina ion o me als on he elec odes made o di e en modi ica ions o ca bon ( ips, ods, glassy ca bon, ca bon pas e wi h di e en con en o ca bon pa icles wi h a ious shapes and sizes) can be ound elsewhe e [45-47]. Based on he ac s men ioned in In oduc ion sec ion, lead(II) ions a e somehow connec ed wi h ca cinogene ic p ocesses, howe e , hei speci ic oles emains unclea . To in es iga e hei oles, some analy ical me hods able o s udy hese ions unde a ious en i onmen s a e needed. The e o e, we p ima ily op imized elec ochemical de e mina ion o hese ions using di e en ial pulse ol amme y. This me hod was op imized o he s anda d solu ion o Pb(NO3)2 wi hin he concen a ion ange o me al ions om 0.2 o 25 ng/ml. Va ious imes o accumula ion (120, 240, 300 and 360 s), pH o suppo ing elec oly e (4.4, 4.6, 4.8, 5.0, 5.2 and 5.4) and deposi ion po en ials (-1.0, -0.8,-0.6, -0.55 and -0.5 V) we e es ed. The ob ained esul s a e shown in Figs. 1A, B and C, espec i ely. The ela i e peak heigh s o lead a e ela ed o he highes alue. I clea ly ollows om he esul s ob ained ha he accumula ion ime o 300 s was he op imal alue o measu e su icien signal du ing easonable ime. As he op imum pH o he ace a e bu e 5.0 was chosen. The highes signal in he op imiza ion o he deposi ion po en ial was achie ed a -0.5 V. The e o e he accumula ion ime o 300 s, pH 5.0 and deposi ion po en ial -0.5 V we e selec ed o he ollowing expe imen s. Fu he , he calib a ion dependence was measu ed. The ob ained concen a ion dependence o lead(II) ions was linea wi hin he ange om 0.2 o 25 ng/ml wi h equa ion o dependence as ollows: y = 0.4173x; R2 = 0.9909, n = 3, RSD = 3.9 % (Fig. 1D). Limi o de ec ion (3 S/N) was es ima ed as 0.07 ng/ml o lead(II) ions. Mo e analy ical pa ame e s a e shown in Table 1. In . J. Elec ochem. Sci., Vol. 9, 2014 4681 Figu e 1. Op imiza ion o elec ochemical de e mina ion o lead. Lead was de e mined by DPV me hod. Op imiza ion o (A) ime o accumula ion, (B) pH, and (C) deposi ion po en ial. The ela i e peak heigh s o lead a e ela ed o he highes alue. (D) Calib a ion cu e o lead de e mined by DPV me hod wi hou mine aliza ion. 0.2 M ace a e bu e (pH=5) was used as an elec oly e. The pa ame e s we e chosen as ollows: ini ial po en ial -0.6 V, end po en ial - 0.2 V, deposi ion po en ial -0.5 V, accumula ion ime 300 s, pulse ampli ude 25 mV, pulse ime 0.04 s, ol age s ep 5.035 mV, ol age s ep ime 0.3 s, sweep a e 0.0168 V/s. 3.2. In e ac ion o lead ions wi h EDTA Due o he ac ha blood sample o pa ien s a e o en collec ed o ubes ha con ain a ious agen s including EDTA, which is commonly used as an an icoagulan [48], he e ec o his complex agen was in es iga ed. EDTA concen a ion o 1.5 - 2 mg pe 1 ml o blood has no signi ican e ec on cell blood coun hus i is ideal o use in haema ology [49]. Pa o his wo k was aimed o in es iga e he e ec o EDTA on he elec ochemical de e mina ion o lead and hei in e ac ion. Figu e 2A shows an elec ochemical signal o EDTA wi hin he concen a ion ange om 6.25 o 250 µM. The elec ochemical signal o EDTA was composed o h ee peaks (E1, E2 and E3) wi h he po en ial a posi ions -0.15 V (E1), 0 V (E2) and 0.15 V (E3), which a e shown in inse s in Fig. 2A. In e ac ion o lead wi h EDTA is shown in Fig. 2B. The e was used he same concen a ion o EDTA (25 µM) o which he g adually inc easing concen a ion o lead wi hin he ange om 0 o 5.5 µg/ml was added. Wi h he inc easing concen a ions o lead he indi idual peaks EDTA dec eased. E3 peak was no de ec ed since he addi ion o lead concen a ion 2.5 µg/ml. A e he addi ion o lead concen a ion 5 µg/ml he signal o lead was de ec ed a he po en ial -0.46 V and he signal o EDTA In . J. Elec ochem. Sci., Vol. 9, 2014 4682 was no de ec ed. In Fig. 2b1, we can see he changes in ela i e lead peak heigh ( ela ed o he lead ions alone) a ec ed by he inc easing concen a ion o EDTA (0.125 – 6.25 µM) wi h he addi ion o lead 12.5 ng/ml. Wi h inc easing concen a ion o EDTA he lead signal dec eased, which con i ms he e ec i eness o EDTA as a chela ing agen . Dec ease in he lead signal (compa ing he lowes (0.125 µM) and highes (6.25 µM) added EDTA concen a ion) was 87 %. Lead signal was no de ec ed when he EDTA concen a ion was highe han 6.25 µM. Figu e 2b2 shows a dec ease in he signal o lead wi h he addi ion o EDTA (25 µM) compa ed o he signal o lead wi hou EDTA (do ed ba s). The ela i e peak heigh s o lead a e ela ed o he highes alue. Figu e 2. In e ac ion o lead ions wi h EDTA. (A) Typical DP ol ammog ams ela ed o he mix u e o lead ions and EDTA. Concen a ion o EDTA a y om 6.25 o 250 µM. The e we e de ec ed h ee peaks E1, E2 and E3. Inse : dependence o indi idual peak po en ials on a ying concen a ion o EDTA. (B) Real ol ammog ams measu ed by in e ac ions o EDTA wi h a ious concen a ions o lead ions. (b1) Dependence o ela i e lead peak heigh ( ela ed o he maximum alue) on concen a ion o EDTA. (b2) Dependence o ela i e lead peak heigh ( ela ed o he maximum alue) on concen a ion o lead. Changes o a ious ela i e peaks heigh s and po en ials wi h a ious concen a ion o lead ions in mix u es o EDTA and lead as ollows: (C) E1 peak, (D) E2 peak, (E) E3 peak and (F) lead peak. 0.2 M ace a e bu e (pH=5) was used as an elec oly e. The pa ame e s we e chosen as ollows: ini ial po en ial -0.6 V, end po en ial 0.2 V, deposi ion po en ial -0.5 V, accumula ion ime 300 s, pulse ampli ude 25 mV, pulse ime 0.04 s, ol age s ep 5.035 mV, ol age s ep ime 0.3 s, sweep a e 0.0168 V/s. In . J. Elec ochem. Sci., Vol. 9, 2014 4683 Table 1. Analy ical pa ame e s o elec ochemical de e mina ion o Cd(II), Zn(II), Pb(II) and Cu(II). Subs ance Reg ession equa ion Linea dynamic ange (μM) Linea dynamic ange (ng/ml) R2a LODb (μM) LOD (ng/ml) LOQc (μM) LOQ (ng/ml) RSDd (%) Cd y = 97.792x 0.007 – 15.00 0.8 – 1686 0.999 0.002 0.2 0.01 1 2.4 Zn y = 108.08x 0.007 – 15.00 0.5 – 981 1.00 0.01 1 0.03 2 6.5 Pb y = 108.52x 0.007 – 15.00 1.5 – 3108 0.999 0.001 0.3 0.01 1 6.6 Cu y = 107.65x 0.007 – 15.00 0.4 – 953 0.999 0.01 1 0.04 2 2.5 a… eg ession coe icien s b…limi s o de ec ion o de ec o (3 S/N) c… limi s o quan i ica ion o de ec o (10 S/N) d… ela i e s anda d de ia ions Figu es 2C-F indica e he ela i e peak heigh s and changes o po en ials o indi idual peaks o EDTA and lead in hei mu ual in e ac ion. Figu e 2C shows he peak E1 o EDTA. Wi h he inc easing concen a ions o lead he peak E1 inc eased and he po en ial alue was shi ed om -0.23 V o -0.06 V. The peak E2 (Fig. 2D) dec eased wi h he inc easing concen a ion o lead and he e was also a shi o he po en ial alue om 0.02 o 0.01 V. Figu e 2E desc ibes he hi d peak o EDTA, which disappea ed a e he second added lead concen a ions ( 2.5 µg/ml). The shi o he po en ial alue was om 0.09 V o 0.1 V. The signal o lead was de ec ed om he added concen a ion o 4.5 µg/ml wi h a cons an po en ial alue o -0.469 V (Fig. 2F). The disappea ance o he peak E3 co esponds o de ec ion o he signal o lead. F om he ob ained esul s he signi ican in e ac ion o lead wi h EDTA is e iden . Nowadays, EDTA is among he widely used an icoagulan s [24] and i s abili y o ac as a chela ing agen should no be neglec ed especially in he case o a po en ial impac o he up ake o me als du ing he blood collec ion and hen ge ing he dis o ed esul s o he ac ual me als amoun in he body. 3.3. Op imiza ion o mic owa e diges ion Fu he , we aimed ou a en ion a sample p epa a ion, which is ano he key o lead(II) de e mina ion. The mos common p ocedu e o sample p epa a ion o me al de e mina ion is i s dissol ing in a ious acids o hei mix u es [50]. De e mina ion o elemen s is o en ealized by we chemis y s ep coupled wi h a sui able de ec ion echnique. We chemis y includes decomposi ion o he sample wi h ypical ino ganic acids (HNO3, HCl, HF, H2SO4, HClO4). Con en ional hea ing o samples was eplaced by he use o mic owa es [51]. Mic owa e hea ing ensu es elimina ion o con amina ion, and i imp o es he speed and e iciency o diges ion o some ypes o samples, which canno be easily sol ed. The p ocess is no only dependen on he used mine al acid and i s olume, bu also on he ime and powe o mic owa es he e o e he op imiza ion is necessa y [52,53]. In ou case mic owa e diges ion was applied o p epa a ion o small amoun o sample acco ding o p o ocol men ioned in he ci ed pape s [27,39]. Pa ame e s o his p ocedu e we e op imized o ob aining he In . J. Elec ochem. Sci., Vol. 9, 2014 4690 28. S. K izko a, O. Zi ka, V. Adam, M. Beklo a, A. Ho na, Z. S obodo a, B. Su es, L. T nko a, L. Zeman and R. Kizek, Czech J. Anim. Sci., 52 (2007) 143. 29. J. Socho , P. Majzlik, P. Salas, V. Adam, L. T nko a, J. Hubalek and R. Kizek, Lis. Cuk o . Repa ., 126 (2010) 414. 30. O. K ys o o a, L. T nko a, V. Adam, J. Zehnalek, J. Hubalek, P. Babula and R. Kizek, Senso s, 10 (2010) 5308. 31. P. Sob o a, L. Vyslouzilo a, O. S epanko a, M. Ry olo a, J. Anyz, L. T nko a, V. Adam, J. Hubalek and R. Kizek, PLoS ONE, 7 (2012) 1. 32. P. Majzlik, A. S asky, V. Adam, M. Nemec, L. T nko a, J. Zehnalek, J. Hubalek, I. P o aznik and R. Kizek, In . J. Elec ochem. Sci., 6 (2011) 2171. 33. A. Klecke o a, P. Sob o a, O. K ys o o a, J. Socho , O. Zi ka, P. Babula, V. Adam, H. Docekalo a and R. Kizek, In . J. Elec ochem. Sci., 6 (2011) 6011. 34. J. Pe lo a, D. Po esil, R. Mikelo a, O. Blas ik, V. Adam, L. T nko a, F. Jelen, R. P usa, J. Kukacka and R. Kizek, Elec ochim. Ac a, 51 (2006) 5112. 35. G. L. Long and J. D. Wine o dne , Anal. Chem., 55 (1983) A712. 36. V. Adam, O. Zi ka, D. Huska, S. K izko a, J. S nadel, V. Ho ak, T. Vaculo ic, K. No o ny, V. Kanicky, J. Kaise and R. Kizek, Febs J., 276 (2009) 96. 37. G. A agay and A. Me koci, Elec ochim. Ac a, 84 (2012) 49. 38. D. Huska, O. Zi ka, V. Adam, M. Beklo a, S. K izko a, L. Zeman, A. Ho na, L. Ha el, J. Zehnalek and R. Kizek, Czech J. Anim. Sci., 52 (2007) 37. 39. D. Hynek, L. K ejco a, S. K izko a, B. Ru kay-Nedecky, J. Pikula, V. Adam, P. Hajko a, L. T nko a, J. Socho , M. Pohanka, J. Hubalek, M. Beklo a, R. V ba and R. Kizek, In . J. Elec ochem. Sci., 7 (2012) 943. 40. M. K emplo a, L. K ejco a, D. Hynek, P. Ba a h, P. Majzlik, V. Ho ak, V. Adam, J. Socho , N. Ce nei, J. Hubalek, R. V ba and R. Kizek, In . J. Elec ochem. Sci., 7 (2012) 5893. 41. S. K izko a, O. K ys o o a, L. T nko a, J. Hubalek, V. Adam, M. Beklo a, A. Ho na, L. Ha el and R. Kizek, Senso s, 9 (2009) 6934. 42. S. K izko a, P. Ryan , O. K ys o o a, V. Adam, M. Galio a, M. Beklo a, P. Babula, J. Kaise , K. No o ny, J. No o ny, M. Liska, R. Malina, J. Zehnalek, J. Hubalek, L. Ha el and R. Kizek, Senso s, 8 (2008) 445. 43. P. Majzlik, J. P asek, L. T nko a, J. Zehnalek, V. Adam, L. Ha el, J. Hubalek and R. Kizek, Lis. Cuk o . Repa ., 126 (2010) 413. 44. J. F. Ping, J. Wu, Y. B. Ying, M. H. Wang, G. Liu and M. Zhang, J. Ag ic. Food Chem., 59 (2011) 4418. 45. I. S anca a, K. Vy as, K. Kalche , A. Walca ius and J. Wang, Elec oanalysis, 21 (2009) 7. 46. I. S anca a, A. Walca ius, K. Kalche and K. Vy as, Cen . Eu . J. Chem., 7 (2009) 598. 47. J. Zima, I. S anca a, J. Ba ek and K. Vy as, C i . Re . Anal. Chem., 39 (2009) 204. 48. H. G. Go dan and N. L. La son, Am. J. Clin. Pa hol., 23 (1955) 613. 49. S. M. Lewis and C. T. S odda , Lab. P ac ., 20 (1971) 787. 50. D. Ca oll and H. C. S a key, Clay Clay Min., 19 (1971) 321. 51. S. Ga cia-Salgado, M. A. Quijano and M. M. Bonilla, Anal. Chim. Ac a, 714 (2012) 38. 52. C. Be iol, L. S ie ano, M. Be elle, F. Del ino and E. A gese, Appl. Geochem., 23 (2008) 1140. 53. V. Sand oni and C. M. M. Smi h, Anal. Chim. Ac a, 468 (2002) 335. 54. D. Fialo a, M. K emplo a, D. Hynek, M. Konecna, J. Kaise , R. Malina, J. Kynicky, O. K ys o o a, R. Kizek and V. Adam, In . J. Elec ochem. Sci., 8 (2013) 7853. 55. M. Konecna and J. Koma ek, Spec oc. Ac a P . B-A om. Spec ., 62 (2007) 283. 56. M. Konecna, J. Koma ek and L. T nko a, Spec oc. Ac a P . B-A om. Spec ., 63 (2008) 700. 57. W. H. Yu, W. J. Lukiw, C. Be ge on, H. B. Niznik and P. E. F ase , B ain Res., 894 (2001) 37. 58. T. Eckschlage , V. Adam, J. H abe a, K. Figo a and R. Kizek, Cu . P o ein Pep . Sci., 10 (2009) 360. In . J. Elec ochem. Sci., Vol. 9, 2014 4691 59. P. Babula, M. Masa ik, V. Adam, T. Eckschlage , M. S ibo o a, L. T nko a, H. Sku ko a, I. P o aznik, J. Hubalek and R. Kizek, Me allomics, 4 (2012) 739. 60. S. K izko a, M. Ry olo a, J. H abe a, V. Adam, M. S ibo o a, T. Eckschlage and R. Kizek, D ug Me ab. Re ., 44 (2012) 287. 61. W. W. Nagel and B. L. Vallee, P oc. Na l. Acad. Sci. U. S. A., 92 (1995) 579. 62. S. E. Theocha is, A. P. Ma geli, J. T. Klijanienko and G. P. Kou aklis, His opa hology, 45 (2004) 103. 63. G. Chu, J. Biol. Chem., 269 (1994) 787. 64. R. P. Pe ez, Eu . J. Cance , 34 (1998) 1535. 65. L. K ejco a, I. Fab ik, D. Hynek, S. K izko a, J. Gumulec, M. Ry olo a, V. Adam, P. Babula, L. T nko a, M. S ibo o a, J. Hubalek, M. Masa ik, H. Binko a, T. Eckschlage and R. Kizek, In . J. Elec ochem. Sci., 7 (2012) 1767. 66. S. K izko a, M. Masa ik, P. Majzlik, J. Kukacka, J. K useo a, V. Adam, R. P usa, T. Eckschlage , M. S ibo o a and R. Kizek, Ac a Biochim. Pol., 57 (2010) 561. 67. M. Namda ghanba i, W. Wobig, S. K ezoski, N. M. Taba abai and D. H. Pe e ing, J. Biol. Ino g. Chem., 16 (2011) 1087. 68. J. Socho , D. Hynek, L. K ejco a, I. Fab ik, S. K izko a, J. Gumulec, V. Adam, P. Babula, L. T nko a, M. S ibo o a, J. Hubalek, M. Masa ik, H. Binko a, T. Eckschlage and R. Kizek, In . J. Elec ochem. Sci., 7 (2012) 2136. 69. J. Socho , M. Pohanka, B. Ru kay-Nedecky, O. Zi ka, D. Hynek, P. Ma es, L. Zeman, V. Adam and R. Kizek, Cen . Eu . J. Chem., 10 (2012) 1442. 70. S. Takahashi, J. Hema ol. Oncol., 5 (2012) 1. 71. A. Vasa ko a, S. K izo a, O. K ys o o a, V. Adam, L. Zeman, M. Beklo a and R. Kizek, Neu oendoc inol. Le ., 30 (2009) 163. 72. A. Zami ska, L. Ma usiak, P. Dziegiel, G. Szybejko-Machaj and J. C. Szepie owski, Pa hol. Oncol. Res., 18 (2012) 849. 73. R. Locigno and V. Cas ono o, In . J. Oncol., 19 (2001) 221. 74. S. Ca elli, A. Ce io i, A. Cabibbo, G. Fassina, M. Ru o and R. Si ia, Science, 277 (1997) 1681. 75. D. M. Townsend, K. D. Tew and H. Tapie o, Biomed. Pha maco he ., 57 (2003) 145. © 2014 The Au ho s. Published by ESG (www.elec ochemsci.o g). This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion license (h p://c ea i ecommons.o g/licenses/by/4.0/).