- Email: [email protected]
Potentiometric stripping analysis of zinc and copper in human teeth and dental materials
ARTICLE IN PRESS
Journal of Trace Elements in Medicine and Biology 22 (2008) 93–99 www.elsevier.de/jtemb
APPLIED METHODOLOGY
Potentiometric stripping analysis of zinc and copper in human teeth and dental materials Biljana M. Kalicˇanina,, Ruzˇica S. Nikolic´b a
Faculty of Medicine, Department of Pharmacy, University of Nisˇ, Bulevar dr ZoranaDjindjic´a 81, Nisˇ 18000, Serbia Faculty of Sciences, Department of Chemistry, University of Nisˇ, Visˇegradska 33, Nisˇ 18000, Serbia
b
Received 6 November 2006; accepted 19 December 2007
Abstract Potentiometric stripping analysis (PSA) with oxygen as the oxidant has been used to determine soluble zinc and copper levels in exfoliated human teeth (all of which required extraction for orthodontic reasons) and commercial dental materials. The soluble zinc and copper contents of teeth were slightly below the zinc and copper contents in whole teeth reported by other researchers, except in the case of tooth with removed amalgam filling. Soluble zinc and copper concentrations of the dental materials and metalceramic crowns were 0.50–6.30, and of 2.00–4.30 mg/g, respectively. The results of this work suggest that PSA may be a good method for zinc and copper leaching studies during the investigation of dental prosthetic materials’ biocompatibility. Corrosive action of acidic media as evidenced by SEM micrographs caused the leaching of metal ions from teeth. r 2008 Elsevier GmbH. All rights reserved. Keywords: PSA; Zinc; Copper; Teeth; Dental prosthetic materials
Introduction Since its introduction in 1976 [1], potentiometric stripping analysis (PSA) has proved to be a very useful technique for the determination of trace metals in various samples [2–6]. Although relatively simple and economic, PSA compares favorably with other methods usually employed for trace metal analysis like atomic absorption spectroscopy (AAS), inductively coupled plasma atomic emission spectroscopy (ICP–AES), and X-ray fluorescence (XRF) [7,8]. One of the common applications of PSA is zinc and copper determination in various matrices, which is very important because their excess amounts in the body can be toxic [7–11].
Corresponding author. Tel.: +381 27326802; fax: +381 18 238770.
E-mail address: [email protected] (B.M. Kalicˇanin). 0946-672X/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.jtemb.2007.12.005
Heavy metals increasingly contribute to the pollution of the environment, playing an important role in the development of human illnesses and toxic effects. Deficiency and excess (toxicity) of these, resulting from exposure to both the natural and manmade environment, can lead to a wide variety of clinical effects. The monitoring of trace element status via tissue sampling has important implications for the identification and correction of such effects. Tissues used for studying exposure to trace elements include blood, urine, fingernails, and hair. Several studies have shown that human teeth are valuable indicators of metal body burden, as well as a readily accessible biological tissue for their analysis [12,13]. Heavy metals are known to substitute for calcium in the hydroxyapatite crystals of dentin, a calcifying tissue that, unlike bone, once formed remodels very slowly [13]. This may be especially important
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for females because it is a potential endogenous source of lead that will be released during pregnancy and lactation [13]. Zinc is an essential element for humans and is homeostatically regulated in the body [14]. It has an important role in protein synthesis and is also a cofactor for many enzymes regulating cell growth and hormone levels, including regulation of gene transcription and growth factor metabolism. Zinc plays an important role in the formation and metabolism of mineralized tissues [15]. Skeletal muscle accounts for approximately 60% of the total body zinc content, and bone, with a zinc concentration of 100–200 mg/g, for approximately 30% [14]. However, excess amounts of zinc in the body can be toxic. Toxicity from increased intake of zinc has been documented following acute inhalation by industrial workers, overdose of oral supplement preparations, and excessive levels in total parenteral nutrition fluids. High-level exposure to zinc compounds can produce respiratory and gastrointestinal toxicity [11]. Copper is an essential trace mineral needed for good health and wellness. Small amounts of copper are essential for life. However, as with all trace minerals, excess amounts of copper in the body can be toxic. The liver and brain contain the largest amounts of copper in the body; other organs contain smaller amounts. As with mercury and lead, high levels of copper are also associated with mental and emotional disorders. Too high level of copper can produce hemolytic anemia, renal insufficiency, diarrhea, and high systolic and diastolic blood pressure [16–20]. According to the literature data, copper and zinc contents range in teeth from 1 to 16 and from 70 to 3500 mg/g [7–10,16,21,29], respectively, which depends on the tooth type, patient’s age, and socio-geographical dwelling conditions. As far as literature is concerned, we found no data about the possibilities of leaching of zinc and copper under the influence of oral fluids and food to which the teeth are daily exposed. Metalceramic crowns that are implanted into a living tissue, due to health or aesthetic reasons, are constantly exposed to corrosive influence of food and drink. Considering the fact that dental implants remain in the human body for a long period of time, it is necessary to check their possible harmful effects upon human health. The adverse effects of the use of dental implants may include possible chronic intoxication with heavy metals. In the present study, the overall contents of zinc and copper, evaporated in 24 h in 4% acetic acid from natural teeth, dental materials and crowns, was determined by PSA with oxygen as oxidant. There are no literature data on Cu and Zn determination with PSA in human teeth or dental prosthetic materials although it was used for Cd and Pb [2].
Material and methods Chemicals Glacial acetic acid (p.a. grade), hydrochloric acid (suprapur grade), standard solution of zinc (1 g/L, Titrisol), standard solution of gallium (1 g/L, Titrisol), standard solution of copper (1 g/L, Titrisol), and standard solution of mercury (1 g/L, Titrisol) were purchased from Merck (Darmstadt, Germany) and have been used as received. Working solutions were prepared by dilution of standard solution with doubly distilled water.
Sample preparation The samples analyzed in this study were: one sample of commercially available dental ceramic (basically an alkaline alumosilicate) (Vita, Germany), two different dyes (Vita, Germany), one metallic porcelain carrier (Bego, Germany), and three metalceramic crowns prepared according to the procedure usually applied in the dental prosthetic practice (crown 1, 2 and 3). The sample preparation procedure was similar to the sample treatment used for lead leaching determination from pharmaceutical glassware [22]. One gram of each sample was treated with 100 mL of 4% acetic acid for 24 h710 min at 2272 1C. An aliquot of 25 mL was analyzed by PSA without further dilution. The teeth analyzed in this study, all permanent teeth of adult persons, were obtained from the University Dental Clinic in Nisˇ . Both caries-free teeth extracted due to orthodontic reasons and teeth with amalgam filling have been investigated. After extraction, the teeth were cleaned with a polyethylene scraper and then rinsed with saline solution and finally with doubly distilled water. Before the treatment with 100 mL of 4% acetic acid, teeth were dried for 6 h at 60 1C and then fractured to small pieces (approx. 0.2–0.3 mm3) with pliers in order to increase accessible surface. However, crouching to powder was avoided in order to prevent complete dissolution of some sensitive teeth structures and because of the contamination risk. The amalgam filling was mechanically removed from teeth, which were then treated in the same way as cariesfree teeth. Natural human teeth and metalceramic crowns used as samples in this study were treated with 4% acetic acid because the oral cavity is usually an acidic environment due to the consumption of acidic foods and various soft drinks. Some experimental studies [23–25] have already shown that the corrosive action of 4% acetic acid causes leaching of ions of heavy metals from natural teeth, metalceramic crowns and dental prosthetic materials.
ARTICLE IN PRESS B.M. Kalicˇanin, R.S Nikolic´ / Journal of Trace Elements in Medicine and Biology 22 (2008) 93–99
Scanning electron microscopy (SEM) was employed for surface morphology analysis of natural teeth. As for other analytical procedures, for SEM analysis extracted teeth were cleaned with a polyethylene scraper and then rinsed with saline solution, then with doubly distilled water and finally with o-xylene. For SEM analysis teeth surface was coated with graphite by vacuum evaporation.
Apparatus and instrumental conditions All the analyses were performed by using a commercially available computerized stripping analyzer (Faculty of Technology, Novi Sad and Elektrouniverzal, Leskovac, Serbia). The analyzer has a program for automatic qualitative and quantitative determination, involving the calculation of element contents. The instrument can be programmed to give deposition potentials between 2 and 2 V and a constant current for the electrolysis or stripping step between 50 and 50 mA, with the parameter setting accuracies DEo2 mV and DIo0.2 mA. The glassy carbon (SIGRADUR-G) working electrode of a total surface area of 7.07 mm2 was pressed into the Teflon tube (outer diameter 8 mm) at an elevated temperature. A Ag/AgCl, KCl (3.5 mol/L) electrode was used as the reference and a platinum wire as the counter electrode. A mechanical Teflon stirrer was also used. A glassy carbon disk working electrode was used as an inert support for the mercury film. Before electrode formation, the glassy carbon surface was swept with filter paper first soaked with acetone and then with doubly distilled water. The mercury film was formed electrolytically from a solution containing 100 mg/L mercury(II)-ions and 0.02 mol/L hydrochloric acid, at a constant current of 50 mA for 240 s. Once deposited, the mercury film could be used for 25–30 analyses. SEM analysis was carried out with a JOEL machine, with energy excitation of 20 keV, with a time of scanning of 100 s.
PSA of zinc and copper We employed the PSA modification with dissolved oxygen as the oxidizing agent assuming diffusion conditions of mass transfer during the analytical step [15]. This PSA modification is the simplest one since it uses already dissolved oxygen as the oxidant, thus reducing the contamination risk arising from the application of some externally added oxidizing agent. The parameters for the PSA determination of zinc and copper in 4% acetic acid, which also served as the supporting electrolyte, had been optimized earlier [26].
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One of the most frequent interferences in PSA is the formation of intermetallic compounds. Copper forms a great number of intermetallic compounds, the most important for PSA being those with Zn, Ga [27]. Formation of Cu–Zn intermetallic compounds is the most frequent interference in PSA due to the presence of both elements in most real samples. One way of eliminating such interferences is the addition of a third element which forms more stable intermetallic compounds with the interfering element. In our study Ga(III) ions were added for zinc determination since Ga–Cu intermetallic compounds are more stable than Cu–Zn compounds. The content of Ga(III) ions was 250 mg/L. Experimental conditions for the Cu and Zn determination are summarized in Table 1. Both the calibration curve and the standard addition methods were used for zinc and copper determination in the acetic acid extracts of dental materials and teeth. For each sample five replicates were performed.
Results and discussion First of all we checked the linearity and the reproducibility of the PSA analytical signal (tox (s)) of zinc and copper in solution. The analytical signal was found to be a linear function of zinc concentration within the range of 5–30 mg/L. The dependence of the PSA analytical signal (tox (s)) on the mass concentration (CZn) follows the equation tOx ¼ 0:330 þ 0:0246C Zn
(1)
For samples with higher content a separate calibration curve was determined and used. The analytical signal was found to be the linear function of copper concentration within the range of 3–20 mg/L. The dependence of the PSA analytical signal (tox (s)) on the mass concentration (CCu) follows the equation tox ¼ 0:4597 þ 0:0272C Cu
(2)
According to the high values of correlation coefficients (r ¼ 0.9976 for zinc and r ¼ 0.9978 for copper) we Table 1. Experimental conditions for the zinc and copper determination by PSA Experimental conditions
Zn
Cu
Deposition potential (V) Final potential (V) Sample volume (mL) Deposition time (s) Resting time (s) Stirring rate (r.p.m.)
1.31 0.15 25 60 15 4000
0.962 0.00 25 300 15 4000
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concluded that there was a very good linearity of PSA analytical signals within the examined zinc and copper concentration range. The values of the soluble zinc and copper of dental prosthetic materials and metalceramic crowns are given in Tables 2 and 3, respectively. The results obtained with the commercial dental materials showed that for a period of 24 h at a temperature of 2272 1C about 1.2 mg/g zinc leached from dental ceramic powder and about 220 mg/g of zinc from metal carrier, during the treatment with 4% acetic Table 2.
acid (pHE2.5). The contents of zinc leaching from metalceramic crowns were the highest of zinc leaching from ceramic powder and dyes. This is probably due to the very high content of zinc in the metal carrier. The largest quantity of Zn is expected to be released from alloys containing noble metals with Zn added for improving their properties [28]. About 2 mg/g of copper from dental ceramic powder, 3 mg/g from cast alloy, and 3–4.5 mg/g from metalceramic crowns leached under the same conditions. Under the same conditions only 3–6 mg/g of zinc was released
The soluble zinc content of dental prosthetic materials and metalceramic crowns (n ¼ 5)
Sample
Calibration curve
Dental ceramic Dye 1 Dye 2 Metal carrier Metalceramic crown 1 Metalceramic crown 2 Metalceramic crown 3
Standard addition
CZn (mg/g)
RSD (%)
CZn (mg/g)
RSD (%)
1.20 0.54 3.34 216 3.75 6.30 4.25
2.83 4.70 3.80 4.39 5.30 7.15 3.20
1.32 0.38 3.50 220.80 2.90 6.69 4.80
5.20 7.10 10.47 7.75 11.30 9.50 6.70
n—number of determinations.
Table 3.
The soluble copper content of dental prosthetic materials and metalceramic crowns (n ¼ 5)
Sample
Calibration curve
Dental ceramic Dye 1 Dye 2 Metal carrier Metalceramic crown 1 Metalceramic crown 2 Metalceramic crown 3
Standard addition
CCu (mg/g)
RSD (%)
CCu (mg/g)
RSD (%)
1.98 2.05 ND 2.61 2.79 3.38 4.30
3.87 1.93 – 2.14 2.90 3.13 3.50
2.01 1.85 ND 2.35 2.40 2.76 4.85
4.32 5.12 – 4.10 12.43 5.93 6.80
n—number of determinations; ND—not detected.
Table 4.
The soluble zinc content of teeth (n ¼ 5)
Sample
Permanent tooth 1 Permanent tooth 2 Permanent tooth 3 Permanent tooth 4 Permanent tooth 5 Tooth with removed amalgam filling 1 Tooth with removed amalgam filling 2 Tooth with removed amalgam filling 3 n—number of determinations.
Calibration curve
Standard addition
CZn (mg/g)
RSD (%)
CZn (mg/g)
RSD (%)
63.70 74.15 84.20 159.40 129.40 565.64 647.30 919.92
5.90 7.40 11.20 8.95 9.86 9.50 11.10 10.30
62.33 71.45 86.37 160.90 132.00 568.87 650.00 921.60
4.80 6.40 10.40 8.90 10.05 11.25 9.90 12.00
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from the metalceramic crowns owing to the various physico-chemical processes during their manufacture, which transformed zinc into the more stable compounds resistant to corrosive action of acids. The values for the soluble zinc content of teeth are given in Table 4, and the values for the soluble copper content of teeth are given in Table 5. From 63.7 to 159 mg/g of Zn was released from permanent teeth during the period of 24 h in 4% acetic acid. Significantly higher quantities of this metal were released from teeth with amalgam fillings (560–920 mg/g), which should be taken into account because these fillings are in the oral cavity for a long period of time. This may be a source of spreading of this metal in the organism causing a toxic effect [7]. From 1 to 3.2 mg/g of copper was released from natural teeth in 4% acetic acid for 24 h. The total amount of Zn and Cu in teeth has been previously reported (70–3500 mg/g for Zn and 1–16 mg/g for Cu [7,8,29]). As it can be expected, the published values of total concentration are higher than the values of soluble Zn and Cu obtained in this work. Significantly higher copper content was released from teeth with amalgam fillings (11–67 mg/g). Increasing content of copper in sealed teeth is the result of metal spread in the tooth-amalgamic seal–oral liquid system. Amalgam fillings may be an important source of Cu(II) ions which – carried by the body fluids – could spread to various tissues and organs [30]. The small difference between the soluble and total zinc and copper content may be indicative of high zinc and copper mobility in surface layers of teeth. The notable exception was the teeth with removed amalgam filling. The much higher soluble zinc and copper contents for this sample could be tentatively assigned to the high zinc and copper contents in dental amalgam. In general, a slightly better reproducibility in the determination of the soluble zinc and copper content was achieved by the calibration curve method, for both dental prosthetic materials and teeth. However, in both cases the PSA method proved to be applicable for the Table 5.
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determination of small amounts of zinc and copper leached from dental prosthetic materials and a great number of analyzed teeth. The soluble metal contents determined in this study were lower than the maximum allowed by the international standards (ISO 7086/2), but it should not be neglected because of the toxic properties of essential metal (Cu, Zn) at high concentrations. SEM micrographs of natural teeth and teeth treated with 4% acetic acid displayed structural changes in teeth caused by the corrosive action of acid solution (Fig. 1). Metal ions were released because of changing the structure of the teeth tissue.
Fig. 1. SEM micrographs of (a) natural tooth and (b) tooth treated with 4% acetic acid.
The soluble copper content of teeth (n ¼ 5)
Sample
Permanent tooth 1 Permanent tooth 2 Permanent tooth 3 Permanent tooth 4 Permanent tooth 5 Tooth with removed amalgam filling 1 Tooth with removed amalgam filling 2 Tooth with removed amalgam filling 3 n—number of determinations.
Calibration curve
Standard addition
CCu (mg/g)
RSD (%)
CCu (mg/g)
RSD (%)
1.01 2.07 2.27 3.20 2.49 66.27 13.99 11.60
1.12 0.00 3.20 2.95 0.87 9.52 8.50 9.13
1.15 2.15 2.37 3.90 2.70 65.85 14.40 10.79
1.80 1.43 2.40 3.55 2.05 10.15 9.54 10.50
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Conclusion The results presented in this work show that significant quantities of Zn and Cu could be released from natural teeth under the influence of 4% acetic acid during 24 h. Significantly higher quantities of these metals were released from teeth with amalgam fillings. These results indicate the possible sources of Cu and Zn ions and the way of their spreading in the organism. The results shown clearly indicate that potentiometric stripping analysis can be successfully applied for zinc and copper leaching studies during the investigation of the dental prosthetic materials’ biocompatibility and human teeth. Both the sensitivity and reproducibility of the PSA method for the analysis of soluble zinc and copper were determined.
Acknowledgment These results are part of projects no. 145072 and 145068, partly financed by the Republic of Serbia Ministry of Science and Environmental Protection.
References [1] Jagner D, Graneli A. Potentiometric stripping analysis. Anal Chim Acta 1976;83:19–26. [2] Noren JG, Hulthe P, Gillberg C. Analysis of lead and cadmium in deciduous teeth by means of potentiometric stripping analysis. Swed Dent J 1987;11:45–52. [3] Jagner D, Sahlin E, Ratana-Ohpas R, Axelsson B. Rapid method for the determination of copper(II) and lead (II) in tap water using a portable potentiometric stripping analyzer. Anal Chim Acta 1993;278:237–42. [4] Ostapczuk P, Eschnauer HR, Scollary GR. Determination of cadmium, lead and copper in wine by potentiometric stripping analysis. Fresenius’ J Anal Chem 1997; 358:723–7. [5] Suturovic´ ZJ, Marjanovic´ NJ, Dostanic´ NM. Potentiometric stripping analysis of lead in vinegars: development of a method. Nahrung 1997;41:111–3. [6] Kalicˇanin BM, Marjanovic´ NJ, Suturovic´ ZJ. Application of potentiometric stripping analysis with constant inverse current in the analytic step for determining lead in glassware. J Serb Chem Soc 2002;67:213–20. [7] Chew LT, Bradley DA, Mohd AY, Jamil MM. Zinc, lead copper in human teeth measured by induced coupled argon plasma atomic emission spectroscopy (ICP-AES). Appl Radiat Isot 2000;53:633–8. [8] Carvalho ML, Marques JP, Marques AF, Casaca C. Evaluation of the diffusion of Mn, Fe, Ba and Pb in middle ages human teeth by synchrotron microprobe Xray fluorescence. Spectrochim Chim Acta B 2007;62: 702–6. [9] Tvinnereim HM, Eide R, Riise T, Fosse G, Wesenberg GR. Zinc in primary teeth from children in Norway. Sci Total Environ 1999;226:201–12.
[10] Malara P, Kwapulinski J, Malara B. Do the levels of selected metals differ significantly between the roots of carious and non-carious teeth? Sci Total Environ 2006;369:59–68. [11] Walsh CT, Sandstead HH, Prasad AS, Newberne PM, Fraker PJ. Zinc: health effects and research priorities for the 1990s. Environ Health Perspect 1994;102:5–46. [12] Nowak B. Occurrence of heavy metals and sodium, potassium and calcium in human teeth. Analyst 1995;120:747–50. [13] Haller LA, Olmez I, Baratz R, Rabinowitz M, Douglas CW. Dentin as a possible bio-epidemiological measure of exposure to mercury. Arch Environ Contam Toxicol 1993;25:124–8. [14] World Health Organization. Trace elements in human nutrition and health, Geneva 1996. [15] Brandao-Neto J, Stefan V, Mendonca BB, Bloise W, Castro AVB. The essential role of zinc in growth. Nutr Res 1995;15:335–58. [16] Ford ES. Serum copper concentration and coronary heart disease among US adults. Am J Epidemiol 2000;151: 1182–8. [17] Marques AF, Marques JP, Casaca C, Carvalho ML. Xray microprobe SRXRF application on human teeth of renal insufficiency patients. Spectrochim Acta B At Spectrosc 2004;59:1675–80. [18] Carvalho ML, Casaca C, Marques JP, Pinheiro T, Cunha AS. Human teeth elemental profiles measured by synchrotron X-ray fluorescence: dietary habits and environmental influence. X-ray Spectrom 2001;30:190–3. [19] Appleton J, Lee KM, Sawicka Kapusta K, Damek M, Cooke M. The heavy metal content of the teeth of the bank vole (Clethrionomys glareolus) as an exposure marker of environmental pollution in Poland. Environ Pollut 2000;110:441–9. [20] Tvinnereim HM, Eide R, Riise T. Heavy metals in human primary teeth: some factors influencing the metal concentrations. Sci Total Environ 2000;255:21–7. [21] Santos Jr. D, Barbosa Jr. F, Souza SS, Krug J. Cryogenic sample grinding for copper, lead and manganese determination in human teeth by slurry sampling GFAAS. J Anal At Spectrom 2003;18:939–45. [22] Kalicˇanin BM, Todorovic´ ZB, Marjanovic´ NJ, Suturovic´ ZJ. Potentiometric stripping analysis of soluble lead in the glassware used for needs of food and pharmaceutical industry. Res J Chem Environ 2001;5:7–12. [23] Kalicˇanin BM, Nikolic´ R, Marjanovic´ N. Application of potentiometric stripping analysis with constant inverse current for determining soluble lead in human teeth. Anal Chim Acta 2004;525:111–4. [24] Nikolic´ R, Kalicˇanin BM, Marjanovic´ NJ. Potentiometric stripping analysis of the soluble lead released from dental ceramic materials. Facta Univ Ser Phys Chem Technol 2001;2:159–63. [25] Nikolic´ R, Kalicˇanin BM, Nikolic´ G. Potentiometric stripping analysis of lead and cadmium leaching from dental prosthetic materials and teeth. J Serb Chem Soc 2004;69:575–80. [26] Nikolic´ R, Kalicˇanin BM, Nikolic´ G. Method development for the potentiometric stripping analysis of soluble
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copper in dental materials. In: International conference on analytical chemistry and chemical analysis, vol. 373, Kyiv, Ukraine; 2005. [27] Tyszczuk K, Korolczuk M, Grabarczyk M. Application of gallium film electrode for elimination of copper interference in anodic stripping voltammetry of zinc. Talanta 2006;71(5):2098–101. [28] Wataha JC, Nelson SK, Lockwood PE. Elemental release from dental casting alloys into biological media with and without protein. Dental Mater 2001;17:409–14.
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[29] Oehme M, Lund W, Jonsen J. The determination of copper, cadmium and zinc in human teeth by anodic stripping voltammetry. Anal Chim Acta 1978;100: 389–98. [30] Sanna G, Piolo MI, Piu PC, Spano N, Tapparo A, Campus GG, et al. Study of the short-term release of the ionic fraction of heavy metals from dental amalgam into synthetic saliva, using anodic stripping voltammetry with microelectrodes. Talanta 2002;58:979–85.
Journal of Trace Elements in Medicine and Biology 22 (2008) 93–99 www.elsevier.de/jtemb
APPLIED METHODOLOGY
Potentiometric stripping analysis of zinc and copper in human teeth and dental materials Biljana M. Kalicˇanina,, Ruzˇica S. Nikolic´b a
Faculty of Medicine, Department of Pharmacy, University of Nisˇ, Bulevar dr ZoranaDjindjic´a 81, Nisˇ 18000, Serbia Faculty of Sciences, Department of Chemistry, University of Nisˇ, Visˇegradska 33, Nisˇ 18000, Serbia
b
Received 6 November 2006; accepted 19 December 2007
Abstract Potentiometric stripping analysis (PSA) with oxygen as the oxidant has been used to determine soluble zinc and copper levels in exfoliated human teeth (all of which required extraction for orthodontic reasons) and commercial dental materials. The soluble zinc and copper contents of teeth were slightly below the zinc and copper contents in whole teeth reported by other researchers, except in the case of tooth with removed amalgam filling. Soluble zinc and copper concentrations of the dental materials and metalceramic crowns were 0.50–6.30, and of 2.00–4.30 mg/g, respectively. The results of this work suggest that PSA may be a good method for zinc and copper leaching studies during the investigation of dental prosthetic materials’ biocompatibility. Corrosive action of acidic media as evidenced by SEM micrographs caused the leaching of metal ions from teeth. r 2008 Elsevier GmbH. All rights reserved. Keywords: PSA; Zinc; Copper; Teeth; Dental prosthetic materials
Introduction Since its introduction in 1976 [1], potentiometric stripping analysis (PSA) has proved to be a very useful technique for the determination of trace metals in various samples [2–6]. Although relatively simple and economic, PSA compares favorably with other methods usually employed for trace metal analysis like atomic absorption spectroscopy (AAS), inductively coupled plasma atomic emission spectroscopy (ICP–AES), and X-ray fluorescence (XRF) [7,8]. One of the common applications of PSA is zinc and copper determination in various matrices, which is very important because their excess amounts in the body can be toxic [7–11].
Corresponding author. Tel.: +381 27326802; fax: +381 18 238770.
E-mail address: [email protected] (B.M. Kalicˇanin). 0946-672X/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.jtemb.2007.12.005
Heavy metals increasingly contribute to the pollution of the environment, playing an important role in the development of human illnesses and toxic effects. Deficiency and excess (toxicity) of these, resulting from exposure to both the natural and manmade environment, can lead to a wide variety of clinical effects. The monitoring of trace element status via tissue sampling has important implications for the identification and correction of such effects. Tissues used for studying exposure to trace elements include blood, urine, fingernails, and hair. Several studies have shown that human teeth are valuable indicators of metal body burden, as well as a readily accessible biological tissue for their analysis [12,13]. Heavy metals are known to substitute for calcium in the hydroxyapatite crystals of dentin, a calcifying tissue that, unlike bone, once formed remodels very slowly [13]. This may be especially important
ARTICLE IN PRESS 94
B.M. Kalicˇanin, R.S Nikolic´ / Journal of Trace Elements in Medicine and Biology 22 (2008) 93–99
for females because it is a potential endogenous source of lead that will be released during pregnancy and lactation [13]. Zinc is an essential element for humans and is homeostatically regulated in the body [14]. It has an important role in protein synthesis and is also a cofactor for many enzymes regulating cell growth and hormone levels, including regulation of gene transcription and growth factor metabolism. Zinc plays an important role in the formation and metabolism of mineralized tissues [15]. Skeletal muscle accounts for approximately 60% of the total body zinc content, and bone, with a zinc concentration of 100–200 mg/g, for approximately 30% [14]. However, excess amounts of zinc in the body can be toxic. Toxicity from increased intake of zinc has been documented following acute inhalation by industrial workers, overdose of oral supplement preparations, and excessive levels in total parenteral nutrition fluids. High-level exposure to zinc compounds can produce respiratory and gastrointestinal toxicity [11]. Copper is an essential trace mineral needed for good health and wellness. Small amounts of copper are essential for life. However, as with all trace minerals, excess amounts of copper in the body can be toxic. The liver and brain contain the largest amounts of copper in the body; other organs contain smaller amounts. As with mercury and lead, high levels of copper are also associated with mental and emotional disorders. Too high level of copper can produce hemolytic anemia, renal insufficiency, diarrhea, and high systolic and diastolic blood pressure [16–20]. According to the literature data, copper and zinc contents range in teeth from 1 to 16 and from 70 to 3500 mg/g [7–10,16,21,29], respectively, which depends on the tooth type, patient’s age, and socio-geographical dwelling conditions. As far as literature is concerned, we found no data about the possibilities of leaching of zinc and copper under the influence of oral fluids and food to which the teeth are daily exposed. Metalceramic crowns that are implanted into a living tissue, due to health or aesthetic reasons, are constantly exposed to corrosive influence of food and drink. Considering the fact that dental implants remain in the human body for a long period of time, it is necessary to check their possible harmful effects upon human health. The adverse effects of the use of dental implants may include possible chronic intoxication with heavy metals. In the present study, the overall contents of zinc and copper, evaporated in 24 h in 4% acetic acid from natural teeth, dental materials and crowns, was determined by PSA with oxygen as oxidant. There are no literature data on Cu and Zn determination with PSA in human teeth or dental prosthetic materials although it was used for Cd and Pb [2].
Material and methods Chemicals Glacial acetic acid (p.a. grade), hydrochloric acid (suprapur grade), standard solution of zinc (1 g/L, Titrisol), standard solution of gallium (1 g/L, Titrisol), standard solution of copper (1 g/L, Titrisol), and standard solution of mercury (1 g/L, Titrisol) were purchased from Merck (Darmstadt, Germany) and have been used as received. Working solutions were prepared by dilution of standard solution with doubly distilled water.
Sample preparation The samples analyzed in this study were: one sample of commercially available dental ceramic (basically an alkaline alumosilicate) (Vita, Germany), two different dyes (Vita, Germany), one metallic porcelain carrier (Bego, Germany), and three metalceramic crowns prepared according to the procedure usually applied in the dental prosthetic practice (crown 1, 2 and 3). The sample preparation procedure was similar to the sample treatment used for lead leaching determination from pharmaceutical glassware [22]. One gram of each sample was treated with 100 mL of 4% acetic acid for 24 h710 min at 2272 1C. An aliquot of 25 mL was analyzed by PSA without further dilution. The teeth analyzed in this study, all permanent teeth of adult persons, were obtained from the University Dental Clinic in Nisˇ . Both caries-free teeth extracted due to orthodontic reasons and teeth with amalgam filling have been investigated. After extraction, the teeth were cleaned with a polyethylene scraper and then rinsed with saline solution and finally with doubly distilled water. Before the treatment with 100 mL of 4% acetic acid, teeth were dried for 6 h at 60 1C and then fractured to small pieces (approx. 0.2–0.3 mm3) with pliers in order to increase accessible surface. However, crouching to powder was avoided in order to prevent complete dissolution of some sensitive teeth structures and because of the contamination risk. The amalgam filling was mechanically removed from teeth, which were then treated in the same way as cariesfree teeth. Natural human teeth and metalceramic crowns used as samples in this study were treated with 4% acetic acid because the oral cavity is usually an acidic environment due to the consumption of acidic foods and various soft drinks. Some experimental studies [23–25] have already shown that the corrosive action of 4% acetic acid causes leaching of ions of heavy metals from natural teeth, metalceramic crowns and dental prosthetic materials.
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Scanning electron microscopy (SEM) was employed for surface morphology analysis of natural teeth. As for other analytical procedures, for SEM analysis extracted teeth were cleaned with a polyethylene scraper and then rinsed with saline solution, then with doubly distilled water and finally with o-xylene. For SEM analysis teeth surface was coated with graphite by vacuum evaporation.
Apparatus and instrumental conditions All the analyses were performed by using a commercially available computerized stripping analyzer (Faculty of Technology, Novi Sad and Elektrouniverzal, Leskovac, Serbia). The analyzer has a program for automatic qualitative and quantitative determination, involving the calculation of element contents. The instrument can be programmed to give deposition potentials between 2 and 2 V and a constant current for the electrolysis or stripping step between 50 and 50 mA, with the parameter setting accuracies DEo2 mV and DIo0.2 mA. The glassy carbon (SIGRADUR-G) working electrode of a total surface area of 7.07 mm2 was pressed into the Teflon tube (outer diameter 8 mm) at an elevated temperature. A Ag/AgCl, KCl (3.5 mol/L) electrode was used as the reference and a platinum wire as the counter electrode. A mechanical Teflon stirrer was also used. A glassy carbon disk working electrode was used as an inert support for the mercury film. Before electrode formation, the glassy carbon surface was swept with filter paper first soaked with acetone and then with doubly distilled water. The mercury film was formed electrolytically from a solution containing 100 mg/L mercury(II)-ions and 0.02 mol/L hydrochloric acid, at a constant current of 50 mA for 240 s. Once deposited, the mercury film could be used for 25–30 analyses. SEM analysis was carried out with a JOEL machine, with energy excitation of 20 keV, with a time of scanning of 100 s.
PSA of zinc and copper We employed the PSA modification with dissolved oxygen as the oxidizing agent assuming diffusion conditions of mass transfer during the analytical step [15]. This PSA modification is the simplest one since it uses already dissolved oxygen as the oxidant, thus reducing the contamination risk arising from the application of some externally added oxidizing agent. The parameters for the PSA determination of zinc and copper in 4% acetic acid, which also served as the supporting electrolyte, had been optimized earlier [26].
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One of the most frequent interferences in PSA is the formation of intermetallic compounds. Copper forms a great number of intermetallic compounds, the most important for PSA being those with Zn, Ga [27]. Formation of Cu–Zn intermetallic compounds is the most frequent interference in PSA due to the presence of both elements in most real samples. One way of eliminating such interferences is the addition of a third element which forms more stable intermetallic compounds with the interfering element. In our study Ga(III) ions were added for zinc determination since Ga–Cu intermetallic compounds are more stable than Cu–Zn compounds. The content of Ga(III) ions was 250 mg/L. Experimental conditions for the Cu and Zn determination are summarized in Table 1. Both the calibration curve and the standard addition methods were used for zinc and copper determination in the acetic acid extracts of dental materials and teeth. For each sample five replicates were performed.
Results and discussion First of all we checked the linearity and the reproducibility of the PSA analytical signal (tox (s)) of zinc and copper in solution. The analytical signal was found to be a linear function of zinc concentration within the range of 5–30 mg/L. The dependence of the PSA analytical signal (tox (s)) on the mass concentration (CZn) follows the equation tOx ¼ 0:330 þ 0:0246C Zn
(1)
For samples with higher content a separate calibration curve was determined and used. The analytical signal was found to be the linear function of copper concentration within the range of 3–20 mg/L. The dependence of the PSA analytical signal (tox (s)) on the mass concentration (CCu) follows the equation tox ¼ 0:4597 þ 0:0272C Cu
(2)
According to the high values of correlation coefficients (r ¼ 0.9976 for zinc and r ¼ 0.9978 for copper) we Table 1. Experimental conditions for the zinc and copper determination by PSA Experimental conditions
Zn
Cu
Deposition potential (V) Final potential (V) Sample volume (mL) Deposition time (s) Resting time (s) Stirring rate (r.p.m.)
1.31 0.15 25 60 15 4000
0.962 0.00 25 300 15 4000
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concluded that there was a very good linearity of PSA analytical signals within the examined zinc and copper concentration range. The values of the soluble zinc and copper of dental prosthetic materials and metalceramic crowns are given in Tables 2 and 3, respectively. The results obtained with the commercial dental materials showed that for a period of 24 h at a temperature of 2272 1C about 1.2 mg/g zinc leached from dental ceramic powder and about 220 mg/g of zinc from metal carrier, during the treatment with 4% acetic Table 2.
acid (pHE2.5). The contents of zinc leaching from metalceramic crowns were the highest of zinc leaching from ceramic powder and dyes. This is probably due to the very high content of zinc in the metal carrier. The largest quantity of Zn is expected to be released from alloys containing noble metals with Zn added for improving their properties [28]. About 2 mg/g of copper from dental ceramic powder, 3 mg/g from cast alloy, and 3–4.5 mg/g from metalceramic crowns leached under the same conditions. Under the same conditions only 3–6 mg/g of zinc was released
The soluble zinc content of dental prosthetic materials and metalceramic crowns (n ¼ 5)
Sample
Calibration curve
Dental ceramic Dye 1 Dye 2 Metal carrier Metalceramic crown 1 Metalceramic crown 2 Metalceramic crown 3
Standard addition
CZn (mg/g)
RSD (%)
CZn (mg/g)
RSD (%)
1.20 0.54 3.34 216 3.75 6.30 4.25
2.83 4.70 3.80 4.39 5.30 7.15 3.20
1.32 0.38 3.50 220.80 2.90 6.69 4.80
5.20 7.10 10.47 7.75 11.30 9.50 6.70
n—number of determinations.
Table 3.
The soluble copper content of dental prosthetic materials and metalceramic crowns (n ¼ 5)
Sample
Calibration curve
Dental ceramic Dye 1 Dye 2 Metal carrier Metalceramic crown 1 Metalceramic crown 2 Metalceramic crown 3
Standard addition
CCu (mg/g)
RSD (%)
CCu (mg/g)
RSD (%)
1.98 2.05 ND 2.61 2.79 3.38 4.30
3.87 1.93 – 2.14 2.90 3.13 3.50
2.01 1.85 ND 2.35 2.40 2.76 4.85
4.32 5.12 – 4.10 12.43 5.93 6.80
n—number of determinations; ND—not detected.
Table 4.
The soluble zinc content of teeth (n ¼ 5)
Sample
Permanent tooth 1 Permanent tooth 2 Permanent tooth 3 Permanent tooth 4 Permanent tooth 5 Tooth with removed amalgam filling 1 Tooth with removed amalgam filling 2 Tooth with removed amalgam filling 3 n—number of determinations.
Calibration curve
Standard addition
CZn (mg/g)
RSD (%)
CZn (mg/g)
RSD (%)
63.70 74.15 84.20 159.40 129.40 565.64 647.30 919.92
5.90 7.40 11.20 8.95 9.86 9.50 11.10 10.30
62.33 71.45 86.37 160.90 132.00 568.87 650.00 921.60
4.80 6.40 10.40 8.90 10.05 11.25 9.90 12.00
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from the metalceramic crowns owing to the various physico-chemical processes during their manufacture, which transformed zinc into the more stable compounds resistant to corrosive action of acids. The values for the soluble zinc content of teeth are given in Table 4, and the values for the soluble copper content of teeth are given in Table 5. From 63.7 to 159 mg/g of Zn was released from permanent teeth during the period of 24 h in 4% acetic acid. Significantly higher quantities of this metal were released from teeth with amalgam fillings (560–920 mg/g), which should be taken into account because these fillings are in the oral cavity for a long period of time. This may be a source of spreading of this metal in the organism causing a toxic effect [7]. From 1 to 3.2 mg/g of copper was released from natural teeth in 4% acetic acid for 24 h. The total amount of Zn and Cu in teeth has been previously reported (70–3500 mg/g for Zn and 1–16 mg/g for Cu [7,8,29]). As it can be expected, the published values of total concentration are higher than the values of soluble Zn and Cu obtained in this work. Significantly higher copper content was released from teeth with amalgam fillings (11–67 mg/g). Increasing content of copper in sealed teeth is the result of metal spread in the tooth-amalgamic seal–oral liquid system. Amalgam fillings may be an important source of Cu(II) ions which – carried by the body fluids – could spread to various tissues and organs [30]. The small difference between the soluble and total zinc and copper content may be indicative of high zinc and copper mobility in surface layers of teeth. The notable exception was the teeth with removed amalgam filling. The much higher soluble zinc and copper contents for this sample could be tentatively assigned to the high zinc and copper contents in dental amalgam. In general, a slightly better reproducibility in the determination of the soluble zinc and copper content was achieved by the calibration curve method, for both dental prosthetic materials and teeth. However, in both cases the PSA method proved to be applicable for the Table 5.
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determination of small amounts of zinc and copper leached from dental prosthetic materials and a great number of analyzed teeth. The soluble metal contents determined in this study were lower than the maximum allowed by the international standards (ISO 7086/2), but it should not be neglected because of the toxic properties of essential metal (Cu, Zn) at high concentrations. SEM micrographs of natural teeth and teeth treated with 4% acetic acid displayed structural changes in teeth caused by the corrosive action of acid solution (Fig. 1). Metal ions were released because of changing the structure of the teeth tissue.
Fig. 1. SEM micrographs of (a) natural tooth and (b) tooth treated with 4% acetic acid.
The soluble copper content of teeth (n ¼ 5)
Sample
Permanent tooth 1 Permanent tooth 2 Permanent tooth 3 Permanent tooth 4 Permanent tooth 5 Tooth with removed amalgam filling 1 Tooth with removed amalgam filling 2 Tooth with removed amalgam filling 3 n—number of determinations.
Calibration curve
Standard addition
CCu (mg/g)
RSD (%)
CCu (mg/g)
RSD (%)
1.01 2.07 2.27 3.20 2.49 66.27 13.99 11.60
1.12 0.00 3.20 2.95 0.87 9.52 8.50 9.13
1.15 2.15 2.37 3.90 2.70 65.85 14.40 10.79
1.80 1.43 2.40 3.55 2.05 10.15 9.54 10.50
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Conclusion The results presented in this work show that significant quantities of Zn and Cu could be released from natural teeth under the influence of 4% acetic acid during 24 h. Significantly higher quantities of these metals were released from teeth with amalgam fillings. These results indicate the possible sources of Cu and Zn ions and the way of their spreading in the organism. The results shown clearly indicate that potentiometric stripping analysis can be successfully applied for zinc and copper leaching studies during the investigation of the dental prosthetic materials’ biocompatibility and human teeth. Both the sensitivity and reproducibility of the PSA method for the analysis of soluble zinc and copper were determined.
Acknowledgment These results are part of projects no. 145072 and 145068, partly financed by the Republic of Serbia Ministry of Science and Environmental Protection.
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