Concordia College Journal of Analytical Chemistry 3 (2012), 33-39
The determination of copper, zinc, and lead in human teeth
using inductively coupled plasma atomic emission
spectrometry (ICP-AES)
Jamie Kern and Laura Mathiason
Department of Chemistry, Concordia College, 901 8th St S, Moorhead, MN 56562
Abstract
The concentrations of copper, lead, and zinc were measured in human teeth of
varying ages. The levels of these metals in the teeth reflect those of the bone. Teeth have
proved to be a considerable source of copper, lead, and zinc, and with the demographical
information of each tooth, general trends were also examined. It was determined that lead
concentration appears to increase with age; however, no such trend was obvious for
copper or zinc. In addition, no significant difference between males and females was
observed.
Introduction
Certain metals are necessary for proper bodily function. For example, the ionic form
of copper is utilized in the electron transport chain as an electron donor.1 In addition, Zn2+
functions to displace electrons forming covalent bonds, usually in amino acid chains, so
that carboxypeptidases can more easily break the bonds. It should be noted that lead does
not appear to have a biological role in the body. It is a heavy metal that is toxic to animals
in small amounts.2
Over time, various ingested elements can build up in the body due to environmental
exposure, diet, and/or disease.3 While elemental deficiency is more commonly a concern,
an excess of trace elements in the body can lead to adverse effects such as high infant
mortality rate and shorter life span.4 Of particular interest in the past has been the study of
lead accumulation in the body. Not only is overexposure to lead toxic, but buildup of this
element can also affect the concentrations of zinc and copper in the body.4
Elements can accumulate in the hard tissues of the body such as bone, teeth, and
fingernails. The most practical of these hard tissues to use for elemental measurements is
teeth because bone is generally not readily available and fingernails are often impure
samples.5 In addition, teeth of different ages can be easily accessed in order to compare the
elemental concentrations of multiple generations of people at one time. Inductively
coupled plasma atomic emission spectrometry (ICP-AES) appears to be the most commonly
used method of determining the concentrations of these trace metals in teeth, but ICP mass
spectrometry,6 particle induced X-ray emission analysis,7 and laser ablation8 have also
been used to determine the concentrations of trace metals in human and animal teeth, as
well as bone. Scientists have determined the ranges of these trace metals in human teeth to
be ≤3 µg/g, 320 ± 10 µg/g,7 and 400-480 µg/g9 for lead, zinc, and copper, respectively. In
this experiment, teeth from a wide range of ages were analyzed for copper, lead, and zinc
concentrations using ICP-AES.
33
Concordia College Journal of Analytical Chemistry 3 (2012), 33-39
Experimental
The method for analyzing teeth was adapted from a previous study by Chew et al.3
Tooth Sample Preparation
Extracted teeth were obtained from people in an age range of 16-83 years. The
extractions were performed by an oral surgeon for functional or cosmetic reasons. Of the
seventeen samples, eight were extracted from Caucasian males, eight from Caucasian
females, and one was from an African American female. Upon receiving the teeth, each was
washed with ultrapure water. Subsequently, each tooth was placed in a 25 mL solution of
concentrated nitric acid (70%) (Fisher) for five minutes to remove leftover tissues. Then,
each tooth was rinsed first with ultrapure water, followed by acetone (analytical grade). In
order to dry the teeth, each was placed in a drying oven for five minutes at 90oC. Once the
teeth had cooled, each was weighed and transferred to a dry test tube, which was filled
with approximately 25 mL of 10% nitric acid (Fisher) solution. The test tubes were then
placed in a water bath at about 90°C for an hour. The samples were then left overnight in a
fume hood. The following day, the test tubes were again placed in the 90oC water bath until
each sample had dissolved. Concentrated nitric acid was added to individual test tubes as
needed to aid the dissolving process, especially in the cases in which the tooth mass was
greater than 2 grams.
Once the samples had dissolved, each solution was filtered into a labeled 50-mL
volumetric flask using hardened, ashless filter paper (Whatman 540). Ultrapure water was
used to rinse each filtrate several times. The solutions were diluted to volume with
ultrapure water.
Standard Solution Preparation
Varying amounts of 1000 ppm Zn (SCP Science), Cu (Ricca), and Pb (Fisher) stock
solutions were diluted to make standard solutions. The ranges used are shown in Table 1.
Table 1. Concentrations of standards used in ICP-AES analysis of tooth samples.
Element
Standard 1
Standard 2
Standard 3
Standard 4
Copper
0.4 ppm
1.0 ppm
1.6 ppm
2.0 ppm
Lead
0.4 ppm
1.0 ppm
1.6 ppm
2.0 ppm
Zinc
1.0 ppm
2.0 ppm
3.0 ppm
4.0 ppm
ICP-AES Analysis
Measurements were made using a Varian 715-ES ICP-AES under the following
conditions: Plasma flow: 15 L/min; Sample uptake: 30 s; Pump rate: 15 rpm; Nebulizer
pressure: 200 kPa; Auxiliary flow: 1.5 L/min. Initially, the machine was aspirated with
ultrapure water. Calibration was completed using the stock solutions. Three replicate
measurements were made at wavelengths of 324.754 nm for Cu, 213.856 nm for Zn, and
220.353 nm for Pb for each sample. Linear equations, calculated from each elemental
34
Concordia College Journal of Analytical Chemistry 3 (2012), 33-39
standard curve, were used in order to determine the elemental concentrations in each
sample from the signal. The uncertainty measurements were calculated by the ICP-AES
software.
Results and discussion
The summary statistics for copper, lead, and zinc concentrations in the analyzed
tooth samples are presented in Table 2. Copper concentrations ranged from 6.04 (±0.01)
µg/g to 57.68 (±0.01) µg/g, with a mean concentration of 17.82 µg/g. In comparison to
Chew et al., these values are higher as the maximum concentration of copper found by
Chew et al. was 6 µg/g. Lead concentrations ranged from 0.373 (±0.005) µg/g to 15.78
(±0.02) µg/g, with a mean concentration of 3.84 µg/g. Finally, zinc concentrations ranged
from 44 (±2) µg/g to 227.23 (±0.02) µg/g, with a mean concentration of 100.49 µg/g tooth.
The values for both lead and zinc are within the normal range measured by Zaichick et al.
as their mean for lead was ≤3 µg/g and the mean for zinc was 320 (±10) µg/g.7 It should be
noted that the high zinc concentrations relative to copper and lead can be explained by
high-protein diets, which are characteristic to many Americans. Beef, pork, and lamb as
well as nuts, whole grains, and legumes are significant sources of zinc in the diet.10
Table 2. Summary statistics of elemental concentrations of copper, lead, and
zinc in teeth samples.
Cu (µg/g)
57.68 (±0.01)
6.04 (±0.01)
13.12
17.82
Tooth
Max
Min
Median
Mean
Pb (µg/g)
15.78 (±0.02)
0.373 (±0.005)
1.45
3.84
Zn (µg/g)
227.23 (±0.02)
44 (±2)
85.56
100.49
In Fig. 1, the elemental concentrations of each tooth sample are shown as a function
of age of the person from whom the tooth was extracted. Lead and zinc concentrations
appear to increase with age while copper concentrations do not appear to depend upon
age. Increased elemental concentrations are expected as age increases; however, diet and
other individual factors could affect the correlation between age and elemental
concentration.
50
40
30
20
10
0
10
60
Age (years)
[Zn] µg/g tooth
[Pb] µg/g tooth
60
[Cu] µg/g tooth
250
20
70
15
10
5
200
150
100
50
0
0
10
60
Age (years)
10
60
Age (years)
Figure 1. Variation of elemental concentrations of copper, lead, and zinc as a function of age.
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Concordia College Journal of Analytical Chemistry 3 (2012), 33-39
The mean concentrations for males and females are compared in Fig. 2. A
significance difference between male and female concentrations was not observed for the
analyzed elements.
40
12
35
10
20
15
10
8
µg/g tooth
25
µg/g tooth
µg/g tooth
30
6
4
2
5
0
0
Copper
Males
180
160
140
120
100
80
60
40
20
0
Zinc
Lead
Females
Males
Males
Females
Females
Figure 2. The mean concentrations of males and females are compared for copper, lead, and zinc. The
error bars represent the standard deviations of each respective set of data.
Age (years)
59
40
26
20
18
13
0
10
20
30
40
50
60
70
[Cu] µg/g tooth
Figure 3. The average concentration of copper is shown for each age tested. A distinct
pattern of increasing concentrations is not obvious.
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Concordia College Journal of Analytical Chemistry 3 (2012), 33-39
Age (years)
79
59
26
20
18
13
0
2
4
6
8
10
12
14
16
18
[Pb] µg/g tooth
Figure 4. The average concentration of lead is shown for each age tested. The
concentrations appear to increase with increasing age.
83
Age (years)
59
26
20
18
13
0
50
100
150
200
250
[Zn] µg/g tooth
Figure 5. The average concentration of zinc is shown for each age tested.
Concentration levels appear to be stable as age increases.
Since sample concentrations of each element were diverse, the average
concentration at each age range was calculated for a more accurate representation of the
correlation of age with concentration. Figures 3 and 5 show the correlation for copper and
zinc, respectively. There is no distinct pattern of increasing concentration with age.
However, a correlation between age and lead concentration is apparent in Fig. 4. According
to Kumagai et al., the lead concentration of teeth increases with age in comparison to other
elements because it has a higher affinity for collagen fibers, a componenet of dentin.5 In
addition, elemental deficiency can be detected by comparing elemental concentrations of
individual teeth samples with accepted values. However, a range of accepted values needs
to be developed by analyzing teeth of subjects with known balanced diets.
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Concordia College Journal of Analytical Chemistry 3 (2012), 33-39
Conclusions
The copper, lead, and zinc concentrations in extracted teeth were determined using
ICP-AES analysis. Significant concentrations were found for each respective element in
every tooth sample, and these ranges of concentrations are not extraordinary in
comparison to previous studies using ICP-AES and other instruments for tooth analysis. In
the age range tested, a trend of increasing average concentrations with increasing age was
observed for lead, but the elemental concentrations of copper and zinc in the teeth show no
such trend. An implication of this study is that elemental deficiency in a human diet can be
detected; hence, future applications of ICP-AES analysis can include the determination of
elemental concentrations in wisdom teeth, which are commonly an uncontaminated source
of trace elements compared to other teeth because of the lack of exposure and dental filling
procedures. These analyses would then signal whether the subject is deficient of any
specific trace elements. Toxic levels of trace elements found can also be determined in the
present teeth analyses. While the present study focuses on the levels of copper, lead, and
zinc in teeth, the concentrations of many other trace elements can be determined as well.
Acknowledgments
We would like to thank Dr. Mark Jensen for his guidance and help throughout the
execution of this experiment. A special thanks for Dr. Ed May and his assistants at Prairie
Oral Surgery in Fargo, North Dakota, is in order. Without him and his staff, the collection of
teeth samples with included demographical information would not have been possible. We
would also like to thank the patients from whom the teeth were extracted.
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