Determination of Fifty Trace Element Contents in Macro and Micro Follicular Colloid Nodular Goiter

Background: Colloid nodular goiter (CNG) is the most common disease of the thyroid, even in non-endemic regions, but an etiology of CNG is unclear. It is known that not only iodine (I) but other trace elements (TE) are involved in goitrogenesis. The present study was performed to clarify the preferential accumulation of some TE either in the colloid or in cells of the thyroid gland. Methods: Fifty TE (Ag, Al, As, Au, B, Be, Bi, Cd, Ce, Co, Cr, Cs, Dy, Er, Eu, Fe, Ga, Gd, Hg, Ho, Ir, La, Li, Lu, Mn, Mo, Nb, Nd, Ni, Pb, Pd, Pr, Pt, Rb, Sb, Sc, Se, Sm, Sn, Tb, Te, Th, Ti, Tl, Tm, U, Y, Yb, Zn, and Zr) in the thyroid tissues with diagnosed CNG were prospectively evaluated in 16 patients with macro-follicular CNG and 13 patients with micro-follicular CNG. Control group included thyroid tissue samples from 105 healthy individuals. Mea- surements were performed using a combination of non-destructive instrumental neutron activation analysis with inductively coupled plasma mass spectrometry. Results: It was found that with a goitrous transformation the Ag, Al, B, Bi, Co, Er, Fe, Hg, Mo, Nd, Ni, Pb, Pr, Rb, Tl, U, Y, and Zn level in thyroid tissue can be significantly changed, and these changes depend on CNG histology. An association between B and Pb content and cell volume of CNG was observed. Conclusion: thyroid, which depend on the histology of goiter.


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 20 th 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][6][7][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][10][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][14][15].
Histologically, the CNG is cellular hyperplasia of the thyroid acini. There are two histological types of CNG: macro-and microfollicular. It is obvious that these two types of CNG have different volume ratios "colloid to cells".

Material and Methods
All patients suffered from СNG (n=29, mean age M±SD was 47±14 years, range  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. 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 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 10 mg (for biopsy) and 50 concerning the INAA-LLR of TE contents in human thyroid, prostate and scalp hair [29,30,50,51]. To determine contents of the TE by comparison with a known standard, biological synthetic standards (BSS) prepared from phenol-formaldehyde resins were used [56]. In addition to BSS, aliquots of commercial, chemically pure compounds were also used as standards. Ten sub-samples of certified reference material A dedicated computer program for INAA-LLR mode optimization was used [57]. 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 were 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.

Results
The comparison of our results for the Ag, Co, Cr, Fe, Hg, Rb, Sb, Se, and Zn mass fractions (mg/kg, dry mass basis) in the normal human thyroid obtained by both INAA-LLR and ICP-MS methods is shown in Table 1.  Table 7.

Effect of Goitrous Transformation on TE Contents
From  (Table 7). 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 B and Pb increasingly associated with thyroid cells.

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, information on B, Bi, Ce, Cs, Er, Ga, La, Li, Nd, Pr, Sb, Sc, Sm, Sn, Tl, and Y content in CNG was not found. Kovalev [58] found elevated levels of Ag in the CNG, but Gudzhedzhiani [59] did not. In study of Kamenev [60] the Al content in goitrous tissue was twice as much as the normal level, but Antonova, et al. [61]  Błazewicz et al. [66], but in the recent study this change was not confirmed [9]. Information on the TE contents in macro-or microfollicular CNG, as well as about the association between TE level and relative volume of colloid and cells in goitrous tissue was not found.