All patients with TMNs (n=41, mean age M±SD was 46±15 years, range 16-75) were hospitalized in the Head and Neck Department of the Medical Radiological Research Centre (MRRC), Obninsk.. 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 trace element contents. In all cases the diagnosis has been confirmed by clinical and morphological results obtained during studies of biopsy and resected materials. Histological conclusions for malignant tumors were: 25 papillary adenocarcinomas, 8 follicular adenocarcinomas, 7 solid carcinomas, and 1 reticulosarcoma. Tissue samples of tumor and visually intact tissue adjacent to tumor were taken from 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. 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 studies were approved by the Ethical Committees of MRRC. 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. Informed consent was obtained from all individual participants included in the study

All tissue samples obtained from tumors and visually intact tissue adjacent to tumors were divided into two portions using a titanium scalpel to prevent contamination by TEs of stainless steel 47. One was used for morphological study while the other was intended for TEs analysis. After the samples intended for TEs analysis were weighed, they were freeze-dried and homogenized 48.

To determine the contents of the TEs by comparison with known data for standard, aliquots of commercial, chemically pure compounds and synthetic reference materials were used 49. Ten subsamples of the Certified Reference Material (CRM) IAEA H-4 (animal muscle) were analyzed to estimate the precision and accuracy of results. The CRM IAEA H-4 subsamples were prepared in the same way as the samples of dry homogenized thyroid tissue.

Details of the relevant facility for EDXRF determination of Cu, Fe, Rb, Sr, and Zn contents with ¹⁰⁹Cd radionuclide source, methods of analysis and the quality control of results were presented in our earlier publications concerning the ¹⁰⁹Cd-EDXRF analysis of human thyroid and prostate tissue 25265051.

Details of the relevant facility for EDXRF determination of I contents with ²⁴¹Am radionuclide source, methods of analysis and the quality control of results were presented in our earlier publication concerning the ²⁴¹Am-EDXRF analysis of human thyroid in norm and pathology 21.

All thyroid samples for TEs analysis were prepared in duplicate, and mean values of TEs contents were used in final calculation. Using Microsoft Office Excel software, a summary of the statistics, including, arithmetic mean, standard deviation of mean, standard error of mean, minimum and maximum values, median, percentiles with 0.025 and 0.975 levels was calculated for TEs contents in nodular and adjacent tissue of thyroids with TMNs. Data for normal thyroid were taken from our previous publications 4243444546. The difference in the results between three groups of samples ( normal , tumor , and adjacent ) was evaluated by the parametric Student s t-test and non-parametric Wilcoxon-Mann-Whitney U-test.

Table 1 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 Cu, Fe, I, Rb, Sr, and Zn mass fraction in “normal”, “tumor”, and “adjacent” groups of thyroid tissue samples.

The ratios of means and the comparison of mean values of Cu, Fe, I, Rb, Sr, and Zn mass fractions in pairs of sample groups such as “normal” and “tumor”, “normal” and “adjacent”, and also “adjacent” and “tumor” are presented in Table 2, Table 3, and Table 4, respectively.

As was shown before 2125265051 good agreement of the Cu, Fe, I, Rb, Sr, and Zn contents in CRM IAEA H-4 samples analyzed by EDXRF with the certified data of this CRM indicates acceptable accuracy of the results obtained in the study of normal , tumor , and adjacent groups of thyroid tissue samples presented in Table 1, Table 2, Table 3, and Table 4

From Table 2, it is observed that in cancerous tissue the mass fraction of I and Zn are 23 times and 25%, respectively, lower whereas mass fractions of Cu and Rb are 3.4 and 1.4 times, respectively, higher than in normal tissues of the thyroid. Thus, if we accept the TEs contents in thyroid glands in the normal group as a norm, we have to conclude that with a malignant transformation the Cu, I, Rb, and Zn in thyroid tissue significantly changed. In a general sense Cu, Fe, and Zn contents found in the normal and adjacent groups of thyroid tissue samples were very similar (Table 3,). However, in the adjacent group mean mass fractions of I and Rb were 1.75 and 2.06 times, respectively, higher, whereas mean value of Sr content 4 times lower than in the normal group. Significant changes of tumor TEs contents in comparison with thyroid tissue adjacent to tumor were found only for I (decrease) and Sr (increase). In malignant tumor Sr contents were approximately 5.4 times higher, while I content 40 times lower than in adjacent group of tissue samples (Table 4). Thus, from obtained results it was possible to conclude that the common characteristics of TMNs in comparison with normal thyroid and visually intact thyroid tissue adjacent to malignant tumors were drastically reduced level of I (Table 2, Table 4).

Characteristically, elevated or reduced levels of TEs observed in thyroid nodules are discussed in terms of their potential role in the initiation and promotion of these thyroid lesions. In other words, using the low or high levels of the TEs in affected thyroid tissues researchers try to determine the role of the deficiency or excess of each TE in the etiology and pathogenesis of thyroid diseases. In our opinion, abnormal levels of many TEs in TMNs could be and cause, and also effect of thyroid tissue transformation. From the results of such kind studies, it is not always possible to decide whether the measured decrease or increase in TEs level in pathologically altered tissue is the reason for alterations or vice versa. According to our opinion, investigation of TEs contents in thyroid tissue adjacent to malignant nodules and comparison obtained results with TEs levels typical of normal thyroid gland may give additional useful information on the topic because these data show conditions of tissue in which TMNs were originated and developed.