Thyroid benign nodules (TBN) are found in two-thirds of the population, which is a serious clinical and social problem worldwide [1]. TBN includes non-neoplastic lesions (various types of thyroid goiter, thyroiditis, and cysts) and neoplastic lesions such as thyroid adenoma. Among TBN, the most common diseases are colloid goiter, thyroiditis, and thyroid adenoma [2-4]. Throughout the 20th century, the prevailing view was that iodine deficiency was the main cause of TBN. However, numerous studies have shown that TBN is a common disease in those countries and regions where the population has never experienced iodine deficiency [4]. Moreover, an excess intake of iodine has also been found to contribute to the occurrence of TBN [5-8]. It also turned out that, along with iodine deficiency and excess, many other dietary, environmental and occupational factors play a role in the etiology of TBN [9-11]. Among these factors, the disruption of the evolutionarily stable intake of many trace elements (TEs) into the human body associated with the industrial revolution is a significant importance [12].

In addition to iodine, which is part of thyroid hormones, and selenium, which is involved in thyroid function, other TEs also perform important physiological functions, such as maintaining and regulating cell function, regulating genes, activating or inhibiting enzymatic reactions, and regulating membrane function [13]. The properties of TEs can be essential or toxic (goitrogenic, mutagenic, carcinogenic) depending on specific tissue needs or tolerance, respectively [13]. Excessive accumulation or imbalance of TEs causes dysfunction of cells and leads to cell degeneration, death, benign or malignant transformation [13-15].

For in vivo and in vitro studies of the content of iodine and other TEs in the normal and pathological thyroid gland, we have developed a set of nuclear analytical and related methods [16-22]. Using this set of methods, the influence of age, gender, and some non-endocrine diseases on the level of iodine in the normal human thyroid gland was studied [23,24]. In addition to iodine, the content of many other thyroidal TEs of apparently healthy men and women was determined. As the results of these studies the age [25-35] and gender dependence of some TEs was revealed [36-41]. In addition, it was found that the content of some TEs of the thyroid gland with colloid goiter, thyroiditis and adenoma differs significantly from the levels of these TEs in the normal thyroid gland [42-45].

In studies of the relationship of TEs in the normal thyroid gland, it was shown that the iodine content almost does not correlate with the content of other TEs. However, the situation changes significantly if, in studies of TEs relationships, not the absolute values of the TEs content are used, but the relative values of iodine/TEs ratios [46,47].

It is generally accepted that the pathogenesis of TBN is multifactorial. The present study was conducted to elucidate the role of TEs relationship disorders in the pathogenesis of TBN. With this in mind, our aim was to evaluate the content of silver (Ag), cobalt (Co), chromium (Cr), iron (Fe), mercury (Hg), iodine (I), rubidium (Rb), antimony (Sb), scandium (Sc), selenium (Se), and zinc (Zn) mass fractions in TBN tissue using instrumental neutron activation analysis (INAA) and calculate individual values of I/TEs ratios. Another aim was to compare the levels of Ag, Co, Cr, Fe, Hg, I, Rb, Sb, Sc, Se, and Zn mass fractions, as well as the levels of I/Ag, I/Co, I/Cr, I/Fe, I/Hg, I/Rb, I/Sb, I/Sc, I/Se, and I/Zn mass fraction ratios in TBN with those in the normal thyroid. Finally, differences in intrathyroidal relationships of I/TEs ratios in normal thyroid and TBN was determined.

The group of patients suffering from TBN (n=79) included persons with colloid nodular goiter (n=46), thyroid adenoma (n=19) and thyroiditis (n=14). All patients with colloid nodular goiter (mean age M±SD was 48±12 years, range 30-64 years), thyroid adenoma (mean age M±SD was 41±11 years, range 22-55 years), and thyroiditis (mean age M±SD was 39±9 years, range 34-50 years) were hospitalized in the Head and Neck Department of the Medical Radiological Research Center. The group of patients with thyroiditis included 8 persons with Hashimoto’s thyroiditis and 6 persons with Riedel’s Struma. Each patient underwent a thick-needle puncture biopsy of thyroid nodules for morphological examination and determination of the TEs content in the obtained material. For all patients the diagnosis was confirmed by clinical and morphological/histological results obtained during studies of biopsy and resected materials.

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. Most of the deaths were caused by trauma incompatible with life. 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 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.

All samples under study were divided into two portions with a titanium scalpel [48]. One was used for morphological study and the other for TEs analysis. Samples intended for TEs analysis were weighed, lyophilized, and homogenized [49]. The mass fraction of TEs was calculated by the relative way of comparing between intensities of corresponding gamma-lines induced by neutrons from nuclear reactor in tissue samples and standards. Aliquots of commercial, chemically pure compounds and synthetic standard materials were used as standards [50]. Ten sub-samples of certified reference material (CRM) IAEA H-4 (animal muscle) and IAEA HH-1 (human hair) were analyzed to evaluate the precision and accuracy of the results. The CRM subsamples were prepared in the same manner as dry homogenized thyroid tissue samples.

Details of sample preparation, activation by neutrons of nuclear reactor, gamma-spectrometry, and quality insurance using CRM IAEA H-4 (animal muscle) and CRM IAEA HH-1 (human hair) were presented in our earlier publications concerning the INAA of TEs contents in human thyroid [18,27-30].

The tissue samples were prepared in duplicate and the average values of the TEs contents were used in the final calculations. Using Microsoft Office Excel software, the main statistical parameters were calculated, including the arithmetic mean, standard deviation, standard error of the mean, minimum and maximum values, median, percentiles with levels of 0.025 and 0.975 for the content of TEs and I/TEs ratios in normal and TBN. The difference in results between normal and TBN was assessed using the parametric Student's t-test and the non-parametric Wilcoxon-Mann-Whitney U-test. Pearson's correlation coefficient was used in Microsoft Office Excel to calculate the relationship between different TEs contents and between different I/TEs content ratios in normal thyroid and TBN.

Table 1 depicts comparison of our data for seven TE in ten sub-samples of CRM IAEA H-4 (animal muscle) and CRM IAEA HH-1 (human hair) with the corresponding certified values of TEs contents in these materials.

Table 2 represents 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 Ag, Co, Cr, Fe, Hg, I, Rb, Sb, Sc, Se, and Zn mass fractions, as well as I/Ag, I/Co, I/Cr, I/Fe, I/Hg, I/Rb, I/Sb, I/Sc, I/Se, and I/Zn mass fraction ratios in normal thyroid and TBN.

The comparison of our results with published data for the Ag, Co, Cr, Fe, Hg, I, Rb, Sb, Sc, Se, and Zn contents in the human thyroid and TBN is shown in Table 3.

Table 4 indicates the differences between mean values of Ag, Co, Cr, Fe, Hg, I, Rb, Sb, Sc, Se, and Zn mass fraction, as well as between mean values of I/Ag, I/Co, I/Cr, I/Fe, I/Hg, I/Rb, I/Sb, I/Sc, I/Se, and I/Zn mass fraction ratios in normal thyroid and TBN estimated using the parametric Student's t-test and the non-parametric Wilcoxon-Mann-Whitney U-test.

The data of inter-thyroidal correlations (values of r – Pearson's coefficient of correlation) between all TEs mass fraction and between I/TEs mass fraction ratios identified by us in normal thyroid and TBN are presented in Table 5.

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).