1. Introduction

Colloid nodular goiter (CNG) is the most common disease of the thyroid, even in non-endemic regions [1]. CNG is clinically detected in about 4% of people older than 30 years [1]. CNG is benign lesion; however, during clinical examination, it can mimic malignant tumors. Furthermore, the origination of CNG can indicate the beginning of malignant transformation of the thyroid gland [2].

Up to now, an etiology of CNG is unclear and probably it is multifactorial [3]. There is opinion that CNG occurs when the thyroid is unable to meet the metabolic demands of the body with sufficient hormone production. The thyroid gland compensates by enlarging, which usually overcomes mild deficiencies of thyroid hormones. For over 20th century, there was the dominant hypothesis that CNG is the simple consequence of iodine (I) deficiency, because I is an essential part of thyroid hormones. However, it was found that CNG is a frequent disease even in those countries and regions where the population is never exposed to I shortage [4]. Moreover, it was shown that I excess has severe consequences on human health and associated with the presence of thyroidal disfunctions and autoimmunity, CNG and diffuse goiter, benign and malignant tumors of gland [5-8]. It was also demonstrated that besides I deficiency and excess many other dietary, environmental, and occupational factors are associated with the CNG incidence [9-11]. Among them a disturbance of evolutionary stable input of many trace elements (TE) in human body after industrial revolution plays a significant role in etiology of thyroidal disorders [12].

Besides I involved in thyroid function, other TE have also essential physiological functions such as maintenance and regulation of cell function, gene regulation, activation or inhibition of enzymatic reactions, and regulation of membrane function [13]. Essential or toxic (goitrogenic, mutagenic, carcinogenic) properties of TE depend on tissue-specific need or tolerance, respectively [13]. Excessive accumulation or an imbalance of the TE may disturb the cell functions and may result in cellular degeneration, death, benign or malignant transformation [13-15].  

In our previous studies the complex of in vivo and in vitro nuclear analytical and related methods was developed and used for the investigation of iodine and other TE contents in the normal and pathological thyroid [16-22]. Iodine level in the normal thyroid was investigated in relation to age, gender and some non-thyroidal diseases [23,24]. After that, variations of TE content with age in the thyroid of males and females were studied and age- and gender-dependence of some TE was observed [25-41]. Furthermore, a significant difference between some TE contents in normal and cancerous thyroid was demonstrated [42-47].

Histologically, the CNG is cellular hyperplasia of the thyroid acini. There are two histological types of CNG: macro- and micro-follicular. It is obvious that these two types of CNG have different volume ratios “colloid to cells”.

The present study was performed to clarify the preferential accumulation of some TE either in the colloid or in cells of the thyroid gland. Having this in mind, our aim was to assess the silver (Ag), cobalt (Co), chromium (Cr), iron (Fe), mercury (Hg), rubidium (Rb), antimony (Sb), scandium (Sc), selenium (Se), and  zinc (Zn) contents in macro- and micro-follicular CNG tissue using instrumental neutron activation analysis with high resolution spectrometry of long-lived radionuclides (INAA-LLR). A further aim was to compare the levels of these TE in the macro- and micro-follicular CNG separately with those in intact (normal) gland of apparently healthy persons, as well as to find differences between the levels of these TE in the macro- and micro-follicular CNG.

All studies were approved by the Ethical Committees of the Medical Radiological Research Centre (MRRC), Obninsk. All the procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments, or with comparable ethical standards.

2. Material and Methods

All patients suffered from СNG (n=29, mean age M±SD was 47±14 years, range 30-64) were hospitalized in the Head and Neck Department of the Medical Radiological Research Centre. Thick-needle puncture biopsy of suspicious nodules of the thyroid was performed for every patient, to permit morphological study of thyroid tissue at these sites and to estimate their TE contents. For all patients the diagnosis has been confirmed by clinical and morphological results obtained during studies of biopsy and resected materials. Histological conclusion for all thyroidal lesions was the macro-follicular CNG (n=16) and micro-follicular CNG (n=13).

Normal thyroids for the control group samples were removed at necropsy from 105 deceased (mean age 44±21 years, range 2-87), who had died suddenly. The majority of deaths were due to trauma. A histological examination in the control group was used to control the age norm conformity, as well as to confirm the absence of micro-nodules and latent cancer.  

All tissue samples were divided into two portions using a titanium scalpel [48]. One was used for morphological study while the other was intended for TE analysis. After the samples intended for TE analysis were weighed, they were freeze-dried and homogenized [49]. The pounded sample weighing about 5-10 mg (for biopsy) and 50 mg (for resected materials) was used for trace element measurement by INAA-LLR. The samples for INAA-LLR were wrapped separately in a high-purity aluminum foil washed with rectified alcohol beforehand and placed in a nitric acid-washed quartz ampoule.

To determine contents of the TE by comparison with a known standard, biological synthetic standards (BSS) prepared from phenol-formaldehyde resins were used [50]. In addition to BSS, aliquots of commercial, chemically pure compounds were also used as standards. Ten certified reference material IAEA H-4 (animal muscle) and IAEA HH-1 (human hair) sub-samples weighing about 50 mg were treated and analyzed in the same conditions that thyroid samples to estimate the precision and accuracy of results.

A vertical channel of nuclear reactor was applied to determine the content of Ag, Co, Cr, Fe, Hg, Rb, Sb, Sc, Se, and Zn by INAA-LLR. The quartz ampoule with samples of thyroid, standards, and certified reference material was soldered, positioned in a transport aluminum container and exposed to a 24-hour neutron irradiation in a vertical channel of the WWR-c research nuclear reactor (Branch of Karpov Institute, Obninsk) with a neutron flux of 1.3×10¹³ n×cm^-2×s^-1. Ten days after irradiation samples were reweighed and repacked.

The samples were measured for period from 10 to 30 days after irradiation. The duration of measurements was from 20 min to 10 hours subject to pulse counting rate. The gamma spectrometer included the 100 cm³ Ge(Li) detector and on-line computer-based MCA system. The spectrometer provided a resolution of 1.9 keV on the ⁶⁰Co 1332 keV line. Details of used nuclear reactions, radionuclides, and gamma-energies were presented in our earlier publications concerning the INAA of TE contents in human prostate and scalp hair [51,52].

A dedicated computer program for INAA mode optimization was used [53]. All thyroid samples were prepared in duplicate, and mean values of TE contents were used in final calculation. Using Microsoft Office Excel, a summary of the statistics, including, arithmetic mean, standard deviation, standard error of mean, minimum and maximum values, median, percentiles with 0.025 and 0.975 levels was calculated for TE contents. The difference in the results between normal thyroid and two groups of CNG (separately macro- and micro-follicular), as well as between two groups of CNG was evaluated by the parametric Student’s t-test and non-parametric Wilcoxon-Mann-Whitney U-test.

3. Results

Table 1 depicts our data for Ag, Co, Cr, Fe, Hg, Rb, Sb, Sc, Se, and Zn mass fractions in ten sub-samples of IAEA H-4 (animal muscle) and IAEA HH-1 (human hair) certified reference material and the certified values of this material.

M – arithmetical mean, SD – standard deviation, a – certified values, b – information values.

Table 1. INAA-LLR data of trace element contents in certified reference material IAEA H-4 (animal muscle) and IAEA HH-1 (human hair) compared to certified values ((mg/kg, dry mass basis)

Table 2 presents certain statistical parameters (arithmetic mean, standard deviation, standard error of mean, minimal and maximal values, median, percentiles with 0.025 and 0.975 levels) of the Br, Cu, Fe, Rb, Sr, Zn mass fraction in normal thyroid (n=105), macro-follicular CNG (n=16), and micro-follicular CNG (n=13).

M – arithmetic mean, SD – standard deviation, SEM – standard error of mean, Min – minimum value, Max – maximum value, P 0.025 – percentile with 0.025 level, P 0.975 – percentile with 0.975 level.

Table 2. Some statistical parameters of Ag, Co, Cr, Fe, Hg, Rb, Sb, Sc, Se, and Zn mass fraction (mg/kg, dry mass basis) in normal thyroid and colloid nodular goiter of different histology (macro- and micro-follicular)

The comparison of Ag, Co, Cr, Fe, Hg, Rb, Sb, Sc, Se, and Zn mass fraction in normal thyroid with those in macro- and micro-follicular CNG is shown in Table 3 and 4, respectively.

The ratios of means and the difference between mean values of Ag, Co, Cr, Fe, Hg, Rb, Sb, Sc, Se, and Zn mass fractions in macro- and micro-follicular CNG are presented in Table 5.

M – arithmetic mean, SEM – standard error of mean, Statistically significant values are in bold.

Table 3. Differences between mean values (M±SEM) of Ag, Co, Cr, Fe, Hg, Rb, Sb, Sc, Se, and Zn mass fraction (mg/kg, dry mass basis) in normal thyroid and macro-follicular colloid nodular goiter

M – arithmetic mean, SEM – standard error of mean, Statistically significant values are in bold.

Table 4. Differences between mean values (M±SEM) of Ag, Co, Cr, Fe, Hg, Rb, Sb, Sc, Se, and Zn mass fraction (mg/kg, dry mass basis) in normal thyroid and micro-follicular colloid nodular goiter

M – arithmetic mean, SEM – standard error of mean, Statistically significant values are in bold.

Table 5. Differences between mean values (M±SEM) of Ag, Co, Cr, Fe, Hg, Rb, Sb, Sc, Se, and Zn mass fraction (mg/kg, dry mass basis) in macro-and micro-follicular colloid nodular goiter

4. Discussion

4.1. Precision and accuracy of results

Good agreement of the Ag, Co, Cr, Fe, Hg, Rb, Sb, Sc, Se, and Zn contents analyzed by INAA-LLR with the certified data of CRM IAEA H-4 and IAEA HH-1 (Table 1) indicates an acceptable accuracy of the results obtained in the study of TE of the thyroid presented in Tables 2–5.

The mean values and all selected statistical parameters were calculated for ten TE (Ag, Co, Cr, Fe, Hg, Rb, Sb, Sc, Se, and Zn) mass fractions (Table 2). The mass fraction of Ag, Co, Cr, Fe, Hg, Rb, Sb, Sc, Se, and Zn were measured in all, or a major portion of normal thyroid and CNG samples.

4.2. Effect of goitrous transformation on TE contents

From Table 3, it is observed that in macro-follicular CNG the mass fraction of Ag, Hg, and Zn is 12.0, 23.3, and 1.36 times, respectively, higher than in tissues of the normal thyroid. From Table 4, it is observed that in micro-follicular CNG the mass fraction of Ag, Co, Hg, Rb, Sc, and Zn is 18.2, 1.86, 20.5, 1.33, 1.91, and 1.48 times, respectively, higher than in tissues of the normal thyroid. Thus, if we accept the TE contents in thyroid glands in the control group as a norm, we have to conclude that with a goitrous transformation the Ag, Co, Hg, Rb, Sc, and Zn level in thyroid tissue can be significantly changed.

4.3.Association between TE levels and relative volume of colloid and cells

Comparison mass fraction of Ag, Co, Cr, Fe, Hg, Rb, Sb, Sc, Se, and Zn in macro- and micro-follicular CNG shown that level of Co in micro-follicular goiter is 40% higher than in macro-follicular goiter (Table 5). Because the relative volume of cells in the micro-follicular CNG is higher than in the macro-follicular CNG, it is possible to conclude that Co increasingly associated with thyroid cells.

4.4. Comparison with published data

The published data on TE contents in the CNG in comparison with normal levels are very scanty and contradictory. For example, Kovalev [54] found elevated levels of Ag in the CNG, but Gudzhedzhiani [55] did not. A significant decrease of the Zn content during goitrous transformation was shown by Błazewicz et al. [56], but in the recent study this change was not confirmed [9].

Information on the TE contents in macro- or micro-follicular CNG, as well as about the association between TE level and relative volume of colloid and cells in goitrous tissue was not found.

4.5. Limitations

This study has several limitations. Firstly, analytical techniques employed in this study measure only ten TE (Ag, Co, Cr, Fe, Hg, Rb, Sb, Sc, Se, and Zn) mass fractions. Future studies should be directed toward using other analytical methods which will extend the list of TE investigated in normal and goitrous thyroid. Secondly, the sample size of macro- or micro-follicular CNG groups was relatively small and prevented investigations of TE contents in CNG group using differentials like gender, stage of disease, and dietary habits of healthy persons and patients with CNG. Lastly, generalization of our results may be limited to Russian population. Despite these limitations, this study provides evidence on goiter-specific tissue of Ag, Co, Hg, Rb, Sc, and Zn level alteration, demonstrates associations between Co content and relative volume of cells in CNG, and shows the necessity to continue TE research of CNG of different histology.

5. Conclusion

In this work, TE analysis was carried out in the tissue samples of normal and goitrous thyroid using INAA-LLR. It was shown that INAA-LLR is an adequate analytical tool for the non-destructive determination of Ag, Co, Cr, Fe, Hg, Rb, Sb, Sc, Se, and Zn content in the tissue samples of human thyroid in norm and pathology, including needle-biopsy cores. It was observed the considerable changes in TE contents in the goitrous transformed tissue of thyroid, which depend on the histology of goiter. It was found that Co predominately accumulates in thyroid cells.

Acknowledgements

The author is extremely grateful to Profs. B.M. Vtyurin and V.S. Medvedev, Medical Radiological Research Center, Obninsk, as well as to Dr. Yu. Choporov, Head of the Forensic Medicine Department of City Hospital, Obninsk, for supplying thyroid samples.

Conflict of Interests

The author declares that there is no conflict of interests regarding the publication of this article.

Funding Sources
None.