Biological Trace Element Research

https://doi.org/10.1007/s12011-020-02542-9

Alteration of Trace Elements in Multinodular Goiter, Thyroid

Adenoma, and Thyroid Cancer
Aleksandar Stojsavljević 1 & Branislav Rovčanin 2,3 & Jovana Jagodić 1 & Đurđa Krstić 1 & Ivan Paunović 2,3 &
Marija Gavrović-Jankulović 1 & Dragan Manojlović 1,4

Received: 16 October 2020 / Accepted: 9 December 2020

# The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021

Abstract
Modest progress has been made in understanding the role of trace elements as endocrine disruptors. The aim of this study was to
examine whether there is a change in the content of trace elements in thyroid disease, as well as whether the ratio of elements
could be considered a blood marker for thyroid disease. In addition, this study examined the influence of biological and clinical/
pathological parameters on the elemental profile. Blood samples from patients diagnosed with multinodular goiter (MNG),
thyroid adenoma (TA), and thyroid cancer (TC) were examined and compared with control samples using chemometric analysis.
The concentrations of essential (Mn, Co, Cu, Zn, Se) and toxic elements (Ni, As, Cd, Pb, U) were determined by ICP-MS. This
study showed for the first time that the content of Mn, Co, Ni, Cu, Zn, Se, and Pb in pathological blood samples was significantly
lower compared to the control, while opposite results were obtained for As, Cd, and U. Based on the classification model, the
most important trace metals for discrimination of MNG and TC from the control group (CG) were Co and Zn, while Co, Zn, and
Mn influenced the distinction of CG from TA. Moreover, it was found that Cu/Zn and U/Se ratios had significantly increased
values in pathological blood samples leading to the possibility of establishing new circulating screening markers. These findings
can represent significant translational information since these diseases are widespread and the diagnostic procedure is still
difficult in many cases.

Keywords Trace elements . Multinodular goiter . Thyroid adenoma . Thyroid cancer

Introduction development of goiter and other thyroid diseases, including

thyroid cancer [3].
Exposure to heavy metals is one of the most important envi- Goiter can be defined as a simple enlargement of the thy-
ronmental factors that adversely affect thyroid homeostasis roid gland. Benign enlargement of the thyroid gland without
[1]. The thyroid gland is highly sensitive to metals, due to its hormonal balance most often occurs as a consequence of io-
ability to accumulate them for a long period of time. As a dine deficiency. In areas with iodine deficiency, this condition
consequence of prolonged exposure to the high content of is described as an endemic goiter [4, 5]. However, iodine
essential and/or toxic metals, endocrine disruption may occur deficiency has not been recognized as a risk factor for the
[2]. Metal trace imbalance may be associated with the formation of solid thyroid nodules [6]. Multinodular goiter
(MNG) can be defined as an enlargement of the thyroid
followed by the presence of multiple nodules in the gland
* Aleksandar Stojsavljević
aleksandars@chem.bg.ac.rs
tissue [7]. The risk factor for the formation of one or more
nodules remains unknown although is probably related to en-
1
vironmental contaminants, besides genetic background and
Department for Analytical Chemistry, Innovation Center, Faculty of
other intrinsic factors [6]. Thyroid adenomas (TA) are benign
Chemistry, University of Belgrade, Studentski trg 12-16,
Belgrade 11000, Serbia thyroid tumors. They are nodular formations localized in the
2 thyroid gland and do not cause infiltration or penetration into
Center for Endocrine Surgery, Clinical Center of Serbia, Koste
Todorovica 8, Belgrade, Serbia its capsule. TA is not an uncommon disease and is present in
3 4% of the adult population [8, 9]. Thyroid cancer (TC) is the
Faculty of Medicine, Clinical Center of Serbia, University of
Belgrade, Belgrade, Serbia most common malignant tumor of the endocrine system, al-
4 though it accounts for less than 1% of all human tumors [10,
South Ural State University, Lenin prospect 76, Chelyabinsk, Russia

11]. Ferley et al. [12] report that in the Serbian population, TC
is at the 24th place from the list of malignant diseases ranked
according to frequency. Slijepcevic et al. [13] mentioned that
the incidence of TC in Serbia in the period 1999–2008 was
3/100.000 (women) and 1/100.000 (men), and the highest
incidence rate was among women between 20 and 29 and
men between 30 and 39 years. The same authors also pointed
out that an increase in newly diagnosed cases of TC in Serbia
can be expected in the coming period. The exact cause of TC
remains unknown. Stojsavljevic et al. [14] were the first to
point out the role of essential/toxic trace metals in the patho-
genesis of TC at the level of thyroid tissue.
Most epidemiological studies have shown that the inci-
dence of various benign and malignant thyroid diseases in
the world is significantly increasing. The incidence rate of
thyroid disease varies greatly between countries due to differ-
ent environmental influences [15]. Therefore, it is necessary to
carefully investigate the influence of trace elements, as one of
the main polluters of the environment and recognized endo-
crine disrupters.
In this regard, this study aimed to examine whether there
are changes in the content of essential and toxic trace elements
in the blood of patients with MNG, TA, and TC, as well as to
examine whether the element ratio could be considered a
blood marker for thyroid disease. This study also aimed to
investigate the influence of biological, clinical, and patholog-
ical parameters on the content of trace elements using univar-
iate and multivariate chemometric analysis. The study design
included a representative number of samples, a valid control
group, a wide range of thyroid diseases (benign MNG disease,
TA as benign tumors, and TC), and an analysis of the most
important trace elements for thyroid pathophysiology using
ICP-MS.
Materials and Methods
Sample Collection
Whole blood samples were collected in the morning hours
after an overnight fast. Approximately 5 mL of blood sample
was collected in trace element free evacuated tubes (BD
Vacutainer) from healthy subjects (n = 65, f/m ratio = 41/24;
mean age: 40 ± 6 years), as well as from patients diagnosed
with MNG (n = 66, f/m ratio = 42/24; mean age: 41 ± 3 years),
TA (n = 44, f/m ratio = 30/14; mean age: 51 ± 4 years), and TC
(n = 66; f/m ratio = 37/29; mean age: 40 ± 6 years). Before the
operation, all blood samples were taken from the patients. The
definitive diagnosis of MNG, TA, and TC, as well as the TC
subtype, was confirmed by pathohistological examination of a
postoperative thyroid tissue sample from each patient. All TC
samples were collected from patients with papillary thyroid
Stojsavljević et al.
carcinoma. Medical professionals made a strict selection of
healthy subjects (blood donors).
In order to avoid intrinsic and extrinsic confounding factors
in the greatest possible extent, persons with a previous history
of any chronic disease such as liver, kidney, cardiac, rheuma-
tologic, neurological, and/or autoimmune disorder, as well as
users who have been on chronic medical therapy or who used
mineral supplements, were excluded from this study. Also,
people working in industries that involve chronic exposure
to heavy metals were not included in the research. Approval
for this research was obtained by the Ethics Committee of
Clinical Centre Serbia, Belgrade (Approval Number:
1575/7). Patients and blood donors voluntarily participated
in this study and written informed consent was obtained from
each subject in accordance with the Declaration of Helsinki.
Sample Preparation and Instrumentation
Nitric acid (65%) and hydrogen peroxide (30%) of a high
grade were supplied by Merck (Darmstadt, Germany).
Concentrated nitric acid was additionally purified using the
Berghof-acid device (BSB-939-IR). Samples were digested
using a microwave digestion system (ETHOS 1, Milestone,
Italy).
Approximately 0.75 g of sample was transferred into a
microwave vessel and the exact weight was measured on an
analytical balance. All samples were digested in a 4:1 (v/v)
mixture of nitric acid (65%) and hydrogen peroxide (30%).
Microwave digestion was performed according to the follow-
ing mode of operation: 5 min heating to 50 °C and heating for
an additional 15 min at 180 °C. After a cooling period,
digested samples were transferred into 25-mL volumetric
flasks and diluted with ultrapure water (resistance of
18.2 MΩ, obtained from Milli Q plus system, Merck). The
blank was prepared in the same way. Freeze-dried standard
reference materials (SRM) of whole blood (SERO210105-
level-1 and SERO210305-level-3) supplied by Seronorm
(Sero AS, Norway) were first reconstituted according to the
manufacturer’s instructions and further prepared as blood
samples.
In order to compensate for matrix-induced ion signal fluc-
tuations and instrumental drift, an internal standard (IS) solu-
tion containing 45Sc at a concentration of 50 μg/L and 71Ga,
89
Y, 115In, 159Tb, and 208Bi at concentrations of 10 μg/L was
equally distributed by a second channel of the peristaltic pump
in the blank, standard solutions, and samples. Diluted IS so-
lution was prepared from stock IS solution (100 mg/L of 7Li,
Sc; 20 mg/L of Bi, Ga, In, Tb, Y) supplied by VHG,
Manchester, UK.
All elements were quantified by inductively coupled
plasma-mass spectrometry, ICP-MS (iCAP Qc, Thermo
Scientific, UK) using external calibration. Pure argon
(99.999%), supplied by Messer (Pančevo, Serbia), was used
Alteration of Trace Elements in Multinodular Goiter, Thyroid Adenoma, and Thyroid Cancer
for plasma formation and dispersion of diluted samples. The
collision cell of ICP-MS was filled with high purity helium
gas (99.999%), also supplied by Messer.
All measurements were performed in the optimized inter-
ference mode, which is based on kinetic energy discrimination
(KED). By applying six calibration solutions in a range from 1
to 250 μg/L, the linearity of the calibration curve for each
element was greater than 0.998. The relative standard devia-
tion (RSD) of three measurings of one sample was < 3%.
Intra-day precision was estimated by calculating the RSD at
three different standard concentrations (10, 50, and 100 μg/L)
in 6 replicates. Inter-day precision was estimated for the same
calibration solutions (6 replicates over 6 consecutive days).
Intra-day and inter-day RSDs were 2.2% and 3.9%, respec-
tively. The limit of detection (LOD) and limit of quantification
(LOQ) were determined according to the mean and standard
deviation of a blank. Tree and ten determinations were made
with a blank solution to set the LOD and LOQ, respectively.
Based on the accuracy obtained with SRMs, the following
isotopes were selected: 55Mn, 59Co, 60Ni, 65Cu, 66Zn, 75As,
82
Se, 111Cd, 208Pb, and 238U. The obtained recovery values for
elements in SRMs were in the range of 90.1‑116.6%.
Statistical Analysis
Descriptive statistics, Kolmogorov Smirnov’s test, and Mann
Whitney U test were performed using the SPSS statistical
software (IBM Statistics 20). In all applied statistical tests,
the significance level was 0.05. Kruskal‑Wallis (KW) test
was performed in a demo version of the NCSS statistical soft-
ware (Number Crucher Statistical System Kaysville, UT,
www.ncss.com). Prior to any multivariate analysis, all data
were auto-scaled. Principal component analysis (PCA) and
partial least square-discriminant analysis (PLS-DA) were per-
formed by PLS ToolBox, v.6.2.1, for MATLAB 7.12.0
(R2011a). The PCA was carried out as an exploratory data
analysis by a singular value decomposition (SVD) algorithm
and 0.95 confidence level for Q and T2 Hotelling limits for the
outliers. PLS-DA was performed by the SIMPLS algorithm
without forcing orthogonal conditions on the model, in order
to condense Y-block variance into the first latent variables and
validated using a venetian blind-validation procedure. The
quality of the models was monitored with the following pa-
rameters: Rcal2 (the cumulative sum of squares of the Ys ex-
plained by all extracted components), RCV2 (the cumulative
fraction of the total variation of Ys that can be predicted by all
extracted components), and Rpred2 (the cumulative fraction of
the total variation of Ys that can be predicted by test compo-
nents) that should be as high as possible, as well as RMSEC
(root mean square errors of calibration), RMSECV (root mean
square errors of cross-validation), and RMSEP (root mean
square errors of prediction) that should be as low as possible.
Results
The content of essential (Mn, Co, Cu, Zn, Se) and toxic trace
elements (Ni, As, Cd, Pb, U), together with the content of
selected ratios (Cu/Zn, Cu/Se, Zn/Se, Zn/Pb, Pb/Se, Pb/Mn,
U/Se, and U/Mn), was examined in blood samples of the
control group (CG), as well as in the group of patients with
thyroid diseases (TC, MNG, and TA). Descriptive statistics
data (mean, standard deviation, minimum and maximum
values, median) are shown in Table 1 and Table 2.
Differences in the content of trace elements between the
groups of CG and thyroid disease (TC, MNG, and TA) were
determined by the KW test. The KW test was used for each
element separately, taking the appropriate variety as one fac-
tor. In cases when the KW test showed a statistically signifi-
cant difference between the medians (P = 0.05), a multiple-
comparison Z-value test was also performed (Table 3). The
content of all analyzed elements and element ratios, except for
Cu/Se, Zn/Pb, and Pb/Mn ratios, was significantly different in
CG compared to thyroid groups (Table 3).
Data were further assorted according to gender, smoking
habits, age, goiter size, and clinicopathological parameters
(the existence of thyroid capsular invasion and intrathyroidal
dissemination in patients with TC). Due to the significant
deviation of the results from the normal distribution, the
Mann-Whitney U test was applied for further analysis
(Tables S1a and S1b, suppl. material). According to gender,
only the Zn content was significantly higher in the blood sam-
ples of the male CG, while the Cu and Cu/Zn content in the
TC and MNG groups was higher in the blood samples of the
females. Smoking habits were found to affect the content of
Cu, Cd, Pb, and Zn/Pb, Pb/Se, and Pb/Mn ratios in CG sam-
ples, as well as the content of Mn and U/Mn ratios in TA
groups and Pb/Mn in MNG groups. In addition, the content
of Cu, Cd, and Pb was significantly increased in CG blood
samples of smokers, while the content of Mn was significantly
increased in TA blood samples of non-smokers. In terms of
age, the groups were divided into two subgroups according to
the average age of the subjects (< 50 vs. > 50 years). CG
samples were found to have significantly increased Zn and
U/Mn content in the group of patients older than 50 years,
while MNG samples had increased Cu/Zn content in the same
group. The difference in goiter size (< 3.5 vs. > 3.5 cm) only
affected the Co content in the MNG group; samples with
goiter size above 3.5 cm had a lower Co content compared
to samples of higher goiter size. The existence of capsular
invasion of the thyroid gland did not show an effect on the
trace element content (P > 0.05), while tumor dissemination
was influenced by the U content.
Patients with TC were further divided into three subgroups
according to the T stage of the malignant tumor (T1, T2, T3)
and three characteristic groups of the N stage of the disease
(No, N1, Nx). KW test showed that there were no significant
 Stojsavljević et al.

Table 1 The summarized parameters of descriptive statistics for ten quantified elements in blood of thyroid patients and control individuals (ng/g)

Mn Co Ni Cu Zn As Se Cd Pb U

CG Mean 14.6 1.88 3.52 1006 5147 0.70 86.1 0.78 23.8 0.05
Median 13.1 1.91 2.82 998 5084 0.66 79.8 0.65 21.2 0.04
St. dev. 7.10 0.58 2.14 246 1130 0.49 29.0 0.57 12.1 0.02
Min 4.50 0.59 1.55 205 3268 0.02 24.8 0.10 6.09 0.01
Max 36.1 2.94 12.4 1468 8194 2.38 157 2.14 47.2 0.11
TC Mean 6.26 0.11 4.89 704 1613 1.16 57.4 1.07 11.4 0.08
Median 4.75 0.04 1.69 659 1570 0.77 60.3 0.66 6.78 0.07
St. dev. 7.05 0.24 9.12 210 376.46 1.03 20.7 1.10 19.2 0.04
Min 0.85 0.003 0.10 171 333.60 0.24 7.79 0.19 0.40 0.04
Max 56.0 1.60 47.3 1723 3016 5.65 101 6.34 148 0.28
MNG Mean 6.38 0.10 3.34 730 1675 1.68 59.0 1.08 9.40 0.08
Median 4.51 0.03 1.25 685 1608 0.98 59.7 0.85 7.48 0.07
St. dev. 6.74 0.28 7.76 144 424 2.90 20.8 0.90 7.66 0.02
Min 2.46 0.002 0.027 517 1122 0.10 9.88 0.18 1.16 0.05
Max 39.1 1.98 54.8 1143 3745 18.5 122 6.06 41.0 0.18
TA Mean 6.47 0.12 6.27 746 1636 1.30 58.5 1.09 10.4 0.08
Median 5.48 0.05 2.05 718 1574 0.83 58.9 0.82 6.59 0.07
St. dev. 4.00 0.20 12.2 193 253 1.75 16.7 0.78 9.37 0.02
Min 2.61 0.03 0.09 506 1079 0.13 18.0 0.21 2.59 0.05
Max 21.0 0.84 52.2 1700 2612 10.3 89.4 2.97 48.1 0.18

Table 2 The summarized

parameters of descriptive Cu/ Cu/ Pb/ Pb/ U/Se U/Mn Zn/ Zn/
statistics for selected metal ratios Zn Se Se Mn Se Pb
in blood of thyroid patients and
control individuals (ng/g) CG Mean 0.20 12.3 0.29 1.97 0.001 0.004 66.3 298
Median 0.21 12.2 0.26 1.53 0.001 0.003 60.9 222
St. dev. 0.06 3.08 0.15 1.33 0.0003 0.002 28.3 201
Min 0.04 5.05 0.09 0.31 0.0001 0.0005 30.6 91.9
Max 0.33 19.1 0.73 6.48 0.002 0.01 200 883
TC Mean 0.45 14.9 0.23 1.96 0.002 0.02 31.9 279
Median 0.41 11.7 0.12 1.48 0.001 0.02 28.2 228
St. dev. 0.16 9.59 0.51 1.72 0.002 0.01 13.5 289
Min 0.26 5.01 0.02 0.17 0.001 0.001 14.8 14.9
Max 1.42 53.2 4.07 9.38 0.01 0.09 87.9 2336
MNG Mean 0.45 14.9 0.23 1.76 0.002 0.02 32.7 268
Median 0.43 11.3 0.12 1.49 0.001 0.01 26.7 221
St. dev. 0.10 9.68 0.51 1.22 0.002 0.01 17.2 237
Min 0.23 6.25 0.02 0.25 0.0004 0.003 12.9 32.5
Max 0.90 59.3 3.88 8.28 0.018 0.04 126 1757
TA Mean 0.46 14.5 0.23 1.83 0.002 0.02 30.9 242
Median 0.46 11.9 0.12 1.42 0.001 0.01 27.1 230
St. dev. 0.08 8.08 0.26 1.36 0.001 0.008 12.6 136
Min 0.30 7.78 0.04 0.21 0.001 0.004 19.1 54.3
Max 0.67 40.3 1.08 5.84 0.01 0.04 72.4 592
Alteration of Trace Elements in Multinodular Goiter, Thyroid Adenoma, and Thyroid Cancer

Table 3 Statistical difference (P) in metal quantity between thyroid and 3a) suggested the existence of two distinctive groups be-
patients and control group (CG) individuals examined by analysis of
longing to CG and corresponding thyroid pathology along the
variance and Kruskal–Wallis test
PC1 direction. The loading plot revealed that the variables Co,
Variable P value LSD (Fisher) Zn, Se, Mn, Cu, and Pb have a strong positive influence on
PC1 (Figs. 1b, 2b, and 3b). The higher content of these ele-
Mn 0.000000 CG (TC, TA, MNG)
ments in the CG affects the separation of these samples from
Co 0.000000 CG (TC, TA, MNG)
pathological ones. U had the greatest negative effects on PC1,
Ni 0.000543 CG (TC, MNG)
while Cd, Ni, and As had a lower effect on classification.
Cu 0.000000 CG (TC, TA, MNG)
Furthermore, these variables were characteristic of each ex-
Zn 0.000000 CG (TC, TA, MNG)
amined group of thyroid diseases (MNG, TA, and TC).
As 0.006789 CG (TC, TA, MNG)
Several blood samples exceeded the limits imposed by the
Se 0.000000 CG (TC, TA, MNG) Hotelling T2 95% probability of an ellipse and could be con-
Cd 0.148753 CG (MNG) sidered outliers.
Pb 0.000000 CG (TC, TA, MNG) In addition to PCA, PLS-DA has been used as a multivar-
U 0.000000 CG (TC, TA, MNG) iate classification method aimed to build mathematical models
Cu/Zn 0.000000 CG (TC, TA, MNG) that can assign each unknown blood sample to an appropriate
Cu/Se 0.711967 / group. Samples from the control and thyroid group of diseases
Zn/Se 0.000000 CG (TC, TA, MNG) were modeled simultaneously using PLS-DA. In addition to
Zn/Pb 0.925019 / the cross-validation procedure and to determine the accuracy
Pb/Se 0.000002 CG (TC, TA, MNG) and sensitivity of the PLS-DA model, the overall data set for
Pb/Mn 0.960768 / CG and TC/MNG/TA was divided into two subsets, a calibra-
U/Se 0.000000 CG (TC, TA, MNG) tion set and a test set, composed of randomly selected samples
U/Mn 0.000000 CG (TC, TA, MNG) for the three models, respectively. The compositions of the
calibration and testing sets, as well as the classification and
validation results, expressed in Rcal2, RCV2, Rpred2, RMSEC,
and RMSECV values, for the three models, are shown in
differences between N stages of TC based on the content of Table 5. Obtained models showed high values of correlation
trace elements, while the high Pb content influenced the dif- coefficients: values of Rcal2 (0.9105–0.9312), RCV2 (0.8510–
ferentiation of T2 from T3 stage (Table S2, suppl. material). 0.9033), and Rpred2 (0.9149–0.9340) as well as low values of
In order to reduce the dimensionality of the data and assess absolute deviations of root mean square error: values of
some trends between the groups of CG, MNG, TA, and TC, as RMSEC between 0.1311 and 0.1148; RMSECV, from
well as to identify important variables, PCA was additionally 0.1545 to 0.1969; and RMSEP, between 0.1441 and 0.1496.
applied. The parameters of the PCA model, the eigenvalues, The absolute differences between RMSECV and RMSEP
and the percentage variances captured by each principal com- were also low (between 0.0077 and 0.0473), showing good
ponent (PC), for the three obtained models, are shown in performances of the PLS-DA models.
Table 4. The mutual projections of the factor scores and their Two latent variables (LVs) were selected due to the
loadings for the first two PCs of the three models are presented smallest RMSECV value for all models. Score plots of data
in Figs. 1, 2, and 3. The score plots of models (Figs. 1a, 2a, for analyzing samples are presented in Figs. 4a, 5a, and 6a.

Table 4 Obtained parameters for PCA models

Model 1 (CG/TC) Model 2 (CG/MNG) Model 3 (CG/TA)

Principal Eigen % variance % variance Eigen % variance % variance Eigen % variance % variance
component value captured by captured value captured by captured value captured by captured
number PC (total) PC (total) PC (total)

1 3.50e+000 34.95 34.95 3.77e+000 37.69 37.69 3.67e+000 36.70 36.70

2 2.29e+000 22.88 57.84 1.78e+000 17.78 55.47 1.52e+000 15.20 51.90
3 1.04 + 000 10.37 68.20 1.19e+000 11.84 67.42 1.16e+000 11.64 63.54
4 1.01e+000 10.11 77.53 1.07e+000 10.70 74.23

Fig. 1 Classification and differentiation of CG and TC samples based on the content of analyzed elements by principal component analysis (PCA). a
Score plot. b Loading plot
Variable importance in the projection (VIP) scores was used
to assess the variables that had the strongest influence on the
differentiation of analyzed groups. The variables that had a
VIP score above 1 are considered significant for the classifi-
cation of blood samples. The most important factors that dis-
tinguished CG from MNG and TC were Co and Zn, while Co,
Zn, and Mn influenced the discrimination of CG from TA
(Figs. 4b, 5b, 6b). A standardized regression coefficient that
reveals the significance of an individual variable in the regres-
sion model is shown in Figs. 4c, 5c, and 6c. The model for
patients with TC showed high positive regression coefficients
for Co, Zn, Mn, Cu, and Pb, as well as negative values for Cd,
U, As, Ni, and Se. The CG model showed identical but the
opposite influence of variables for all analyzed models (Figs.
4d, 5d, 6d). Statistical parameters of the PLS-DA model
(Table 5) confirmed the good predictive power of the pro-
posed relations. Score plots of the data for samples also
Fig. 2 Classification and differentiation of CG and MNG samples based on the content of analyzed elements by principal component analysis (PCA). a
Score plot. b Loading plot
Stojsavljević et al.
confirmed the good predictive ability of PLS-DA modeling.
Therefore, the elemental content data enables accurate predic-
tion of thyroid disease and provides a useful tool for discrim-
inating the thyroid group of patients from the healthy group.
Discussion and Conclusion
Considering that the high content of essential trace elements
or even a small amount of metal can be toxic to the thyroid
gland, due to its carcinogenic, mutagenic, and/or deregulatory
nature, the relationship between trace elements and the occur-
rence of neoplastic and other thyroid diseases should be care-
fully examined [16, 17].
This study showed that the content of Mn, Co, Ni, Cu, Zn,
Se, and Pb was significantly reduced in pathological samples
of the thyroid gland compared to CG, while the contents of
Alteration of Trace Elements in Multinodular Goiter, Thyroid Adenoma, and Thyroid Cancer

Fig. 3 Classification and differentiation of CG and TA samples based on the content of analyzed elements by principal component analysis (PCA). a
Score plot. b Loading plot

As, Cd, and U were opposite (P < 0.05). Moreover, it was Comparing our results with other papers, similarities and
found that the Cu/Zn and U/Se ratios in pathological thyroid contradictions were found in the profiles of the trace elements.
samples were significantly higher than the control ones, which The greatest similarity in the examination of the element pro-
could be significant from a clinical point of view (e.g., diag- files was found for Se status. Federige et al. [22] mentioned
nostic purposes, rapid screening). that decreased Se content may be associated with an increased
The first clinical evidence that severe Se deficiency could risk of thyroid disease. A paper by Fiore et al. [23] points out
be detrimental to thyroid health has been found in central that Se could be involved in the development of TC, as well as
Africa. However, well-described diseases associated with se- that the prevalence of an enlarged thyroid gland is significant-
vere Se deficiency, Keshan, and Kashin-Beck disease were ly lower at an adequate level of Se than that in the low Se
not associated with thyroid dysfunction [18]. In addition to category. Moncaio et al. [24] found that patients with TC had
some Chinese regions, Serbia is among the countries with significantly reduced Se values. The study by Kohrle et al.
reported low Se content in real samples [19]. Our latest re- [25] has shown that decreased serum Se levels are highly
search indicated a lack of Se in whole blood and serum sam- correlated with an increase in the incidence of TC in such
ples of the Serbian population [20], as well as in healthy thy- patients, who are at increased risk for developing TC.
roid tissues collected from different groups of subjects [21]. For other essential trace metals, such as Zn and Cu, incon-
Based on the results of this study, patients with TC have a sistent results have been reported between studies. For exam-
significantly low Se content compared to CG. Moreover, the ple, Gumulec et al. [26] did not find a significant difference
values for Se in the CG are lower compared to other popula- between CG and TC using the method of random effects in the
tions worldwide, indicating that even healthy people may be at meta-analysis. In contrast, Al-Sayer et al. [3] found that the
risk of developing Se deficiency. serum Zn content in TC was significantly reduced compared
to the healthy one and that surgical excision of the malignant
thyroid tissue resulted in the restoration of the Zn content in
Table 5 Obtained statistical performance for PLS-DA models normal values. Our results can confirm their finding since Zn
CG/TC CG/MNG CG/TA was measured in samples collected before surgery. Al-Sayer
et al. [3] also reported that the Cu content in the sera of patients
Rcal2 0.9268 0.9312 0.9105 with TC did not differ from the control ones, although the
RCV2 0.9033 0.8510 0.8866 postoperative Cu content was significantly elevated in TC.
Rpred2 0.9340 0.9149 0.9297 Kosova et al. [27] reported increased Cu content in women
RMSEC 0.1343 0.1311 0.1448 with TC before and after surgery. Przybylik-Mazurek et al.
RMSECV 0.1545 0.1969 0,1636 [28] showed that the group of patients with TC had a higher
RMSEP 0.1468 0.1496 0.1441 Cu compared to the CG. Kucharzewski et al. [29] mentioned
Calibration set 40/51 40/51 40/24 that the highest level of Cu and Zn, as well as the ratios Cu/Zn,
Testing test 15/15 15/15 15/10 Cu/Se, and Zn/Se in the whole blood of patients with TC, may
suggest the progression of the proliferation process in the

Fig. 4 PLS-DA performed on data of element content of CG and TC samples. a Score plots of data. b Plots of the variables vs. VIP scores. c and d Plot of
the coefficients of parameters
thyroid gland. Due to discrepancies in the type of blood ma-
terials (whole blood, serum, plasma), it is difficult to make an
appropriate comparative analysis of our results with the re-
ported values. Our results showed that the Zn content was
significantly higher, while the Cu content was significantly
lower in patients with TC. In addition, this study showed that
the Cu/Zn ratio can be considered an appropriate blood marker
for the separation of TC from healthy subjects (Table 2).
Based on our best knowledge, the blood status of other
essential trace metals is unknown in the literature. Al-Sayer
et al. [3] reported only decreased serum Mn levels after the
removal of thyroid malignant tissue. We found that the con-
tent of Mn in whole blood is higher in CG than in TC. Our
study also showed that there is one interesting finding related
to the content of Co. Its values were significantly lower in
patients with TC, as well as in other examined thyroid diseases
compared to CG (Table 2).
The status of toxic trace metals is even less known than
that of essential trace elements. Petrosino et al. [30] found
Stojsavljević et al.
that patients with head and neck cancer (including several
TC blood samples) had at least twice the level of heavy
metals in their blood above the maximum reference level.
Rezaei et al. [16] found that toxic trace metals (Pb and
Cd) may increase the risk of developing TC. We found
that there are significant differences in the content of toxic
metals (Ni, As, Pb, Cd, and U). Since the contents of Ni
and Pb are higher in CG, the relevance of these two toxic
trace metals is of minor importance. However, the in-
creased content of As, Cd, and U in the blood samples
of TC patients deserves further research.
Kravchenko et al. [31] found that patients with nodular
goiter (NG) have a lower serum Cu, Cu, and Zn content
compared to controls, and that lower Se, Cu, and Zn con-
tents may lead to a higher risk of developing NG. In con-
trast, Liu et al. [32] point out that the level of circulating Se
is not recognized as a risk factor for goiter, and that its role
in determining thyroid volume is of minor importance.
Kazi et al. [33] found increased Cu in goitrous patients
Alteration of Trace Elements in Multinodular Goiter, Thyroid Adenoma, and Thyroid Cancer
Fig. 5 PLS-DA performed on data of element content of CG and MNG samples. a Score plots of data. b Plots of the variables vs. VIP scores. c and d Plot
of the coefficients of parameters
compared with CG. Błazewicz et al. [2] showed that Cu,
Mn, and Zn blood levels were significantly increased in
healthy thyroids compared to NG. Furthermore, no statis-
tical differences for Co were found between the groups.
Interestingly, Cd was detected only in NG samples, while
Ni was not found in either healthy or NG samples. Giray
et al. [34] state that the contents of Se and Zn in plasma do
not differ from the control ones, while for Cu and Mn, it
was found that the contents are significantly higher in pa-
tients with MNG. Fiore et al. [23] noted that Pb levels were
increased in TA and decreased in NG compared to TC.
Derumeaux et al. [35] mentioned the protective effect of
Se against goiter and thyroid tissue damage. Due to dis-
agreement on the type of blood material (whole blood,
serum, plasma), it is difficult to make a clear comparative
analysis with the reported values. The results of this study
show that patients with MNG and TA had a significantly
reduced content of Mn, Co, Ni, Cu, Zn, Se, and Pb, as well
as increased content of As, Cd, and U compared to control
blood samples (Table 1).
The results obtained in this study clearly show the presence
of altered contents of toxic/essential trace elements in the
blood of patients with MNG, TA, and TC. This study showed
for the first time that the content of Mn, Co, Ni, Cu, Zn, Se,
and Pb was significantly lower in pathological blood samples
compared to control, while opposite results were obtained for
As, Cd, and U. PCA and PLS-DA have been successfully
used to distinguish pathological thyroid samples from control
ones. In addition, PCA and PLS-DA could be used as a tool to
classify unknown samples. The most important trace elements
that distinguished TC and MNG from CG were Co and Zn,
while Co, Zn, and Mn influenced the discrimination of CG
from TA. This study also showed that Cu/Zn and U/Se ratios
can be considered appropriate markers for screening, as well
as for distinguishing thyroid patients from individuals without
thyroid disease. These findings can represent significant

Fig. 6 PLS-DA performed on data of element content of CG and TA samples. a Score plots of data. b Plots of the variables vs. VIP scores. c and d Plot of
the coefficients of parameters
translational information since these diseases are widespread
and the diagnostic procedure is still difficult in many cases.
Supplementary Information The online version contains supplementary
material available at https://doi.org/10.1007/s12011-020-02542-9.
Funding This research was financially supported by the Ministry of
Education, Science and Technological Development of Republic of
Serbia contract number: 451-03-68/2020-14/200288.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of
interest.
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