COMPARISON BETWEEN BROMINE, CALCIUM, CHLORINE, IODINE, POTASSIUM, MAGNESIUM, MANGANESE, AND SODIUM CONTENTS IN MACRO- AND MICRO-FOLLICULAR COLLOID GOITER

Objective: Colloid nodular goiter (CNG) is the most common disease of the thyroid, even in non-endemic regions, but the etiology of CNG is unclear. It is known that not merely iodine (I) but other chemical elements (ChE) are involved in goitrogenesis. The current study was performed to clarify the preferential accumulation of some ChE either in the colloid or in cells of the thyroid gland.
Methods: Eight ChE: Bromine, calcium, chlorine (Cl), I, potassium, magnesium, manganese, and sodium (Na) in the thyroid tissues with diagnosed CNG were prospectively evaluated in 16 patients with macrofollicular CNG and 13 patients with microfollicular CNG. The control group included thyroid tissue samples from 105 healthy individuals. Measurements were conducted using non-destructive instrumental neutron activation analysis with high-resolution spectrometry of short-lived radionuclides.
Results: It was found that in macrofollicular CNG, the mass fraction of Cl and Na was 2.57 and 1.82 times, respectively, higher than in tissues of the normal thyroid. In microfollicular CNG, the mass fraction of I was 59% lower, whereas the mass fraction of Na was 67% higher than in tissues of the normal thyroid. The level of I in macrofollicular goiter was 2.08 times higher than in microfollicular goiter
Conclusion: There are substantial changes in ChE contents in the goitrous transformed tissue of the thyroid, which depend on the histology of the goiter.


INTRODUCTION
Colloid nodular goiter (CNG) is the most common thyroid disease, even in non-endemic regions [1]. CNG is clinically identified in about 4% of people older than 30 years [1]. CNG is a benign lesion; however, during clinical examination, it can imitate malignant tumors. Furthermore, the origination of CNG can stipulate the beginning of the malignant transformation of the thyroid gland [2]. Up to now, the etiology of CNG is unclear, and it is probably multifactorial [3]. There is an opinion that CNG occurs when the thyroid is not able to meet the metabolic demands of the body with adequate hormone production. The thyroid gland compensates by enlarging, which usually overcomes mild deficiencies of thyroid hormones. For over the 20 th century, there was the governing opinion that NG is the straightforward sequel of iodine (I) deficiency. Although, it was found that NG is a frequent disease even in those countries and regions where the inhabitants are never exposed to I shortage [4]. Moreover, it was found that I excess has severe effects on human health and is associated with the presence of thyroidal dysfunctions and autoimmunity, NG and diffuse goiter, benign and malignant tumors of the gland [5][6][7][8]. It was also demonstrated that besides the I deficiency and excess, many other dietary, environmental, and occupational factors are associated with the NG incidence [9][10][11]. Among them, a disruption of evolutionary stable input of many chemical elements (ChE) in the human body after the industrial revolution plays a significant role in the etiology of thyroidal disorders [12].
In addition to I, many other ChE is involved in essential physiological functions [13]. Crucial or toxic (goitrogenic, mutagenic, and carcinogenic) properties of ChE depend on tissue-specific need or tolerance, respectively [13]. Deficiency, overload, or an imbalance of the ChE may result in cellular dysfunction, degeneration, death, and 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 micro-follicular. It is clear that these two types of CNG have different volume ratios, "colloid to cells." The present study was executed to elucidate the preferential accumulation of some ChE either in the colloid or in cells of the thyroid gland. Having this in mind, we focused on assessing the bromine (Br), calcium (Ca), chlorine (Cl), I, potassium (K), magnesium (Mg), manganese (Mn), and sodium (Na) contents in macro-and microfollicular CNG tissue using non-destructive instrumental neutron activation analysis with high-resolution spectrometry of short-lived radionuclides (INAA-SLR). A further objective was to compare the levels of these ChE 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 ChE in the macro-and micro-follicular CNG.

METHODS
All patients who 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 MRRC. A 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 ChE contents. The diagnosis has been confirmed for all patients by clinical and morphological results acquired throughout studies of biopsy and resected materials. The histological conclusion for all thyroidal lesions was the macrofollicular CNG (n=16) and microfollicular CNG (n=13).
Normal thyroids for the control group samples were drawn out 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, also to confirm the absence of micronodules and latent cancer.
All tissue samples were divided into two parts using a titanium scalpel [48]. One was used for morphological study, while the other was for ChE analysis. After the samples intended for ChE analysis were weighed, they were freeze-dried and homogenized [49]. The pounded samples weighing about 10 mg (for biopsy) and 100 mg (for resected materials) were used for ChE measurement by INAA-SLR.
Details of sample preparation, activation by neutrons of nuclear reactor, gamma-spectrometry, calibration with biological synthetic standards, and quality insurance using certified reference material (CRM) of International Atomic Energy Agency IAEA H-4 (animal muscle) were presented in our earlier publications concerning the INAA-SLR of ChE contents in human thyroid, scalp hair, and prostate [18,27,28,50].
A dedicated computer program for INAA-SLR mode optimization was used [51]. All the thyroid samples were prepared in duplicate, and mean values of ChE contents were used in the final calculation. Using Microsoft Office Excel, a summary of the statistics, including arithmetic mean, standard deviation, standard error of the mean, minimum and maximum values, median, and percentiles with 0.025 and 0.975 levels, was calculated for ChE contents. The distinction in the results between normal thyroid and two groups of CNG (separately macro-and microfollicular), 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. Table 1 presents certain statistical parameters (arithmetic mean, standard deviation, standard error of the mean, minimal and maximal values, median, and percentiles with 0.025 and 0.975 levels) of the Br, Ca, Cl, I, K, Mg, Mn, and Na mass fraction in normal thyroid (n=105), macrofollicular CNG (n=16), and microfollicular CNG (n=13).

IRESULTS
The comparison of Br, Ca, Cl, I, K, Mg, Mn, and Na mass fraction in normal thyroid with those in macro-and micro-follicular CNG is shown in Tables 2 and 3, respectively.
The ratios of means and the distinction between mean values of Br, Ca, Cl, I, K, Mg, Mn, and Na mass fractions in macro-and micro-follicular CNG are presented in Table 4.

Precision and accuracy of results
Previously found good agreement of the Br, Ca, Cl, I, K, Mg, Mn, and Na contents analyzed by INAA-SLR with the certified data of CRM IAEA H-4 [18,27,28,50] indicates an acceptable accuracy of the results obtained in the study of ChE of the thyroid samples presented in Tables 1-4. The mean values and all chosen statistical parameters were calculated for eight ChE (Br, Ca, Cl, I, K, Mg, Mn, and Na) mass fractions ( Table 1). The mass fraction of Br, Ca, Cl, I, K, Mg, Mn, and Na was measured in all or a major portion of normal thyroid and CNG samples.

Effect of goitrous transformation on ChE contents
From Table 2, it is observed that in macrofollicular CNG, the mass fraction of Cl and Na is 2.57 and 1.82 times, respectively, higher than    in tissues of the normal thyroid. From Table 3, it is observed that in microfollicular CNG, the mass fraction of I is 59% lower, whereas the mass fraction of Na is 67% higher than in tissues of the normal thyroid. Thus, if we accept the ChE contents in thyroid glands in the control group as a norm, we have to conclude that the Cl, I, and Na level in thyroid tissue can be notably changed with a goitrous transformation.

Association between ChE levels and relative volume of colloid and cells
Comparison mass fraction of Br, Ca, Cl, I, K, Mg, Mn, and Na in macro-and micro-follicular CNG shown that level of I in macrofollicular goiter is 2.08 times higher than in microfollicular goiter (Table 4). Because the relative volume of colloid in the macrofollicular CNG is higher than in the microfollicular CNG, it is possible to conclude that I increasingly associated with colloid.

Comparison with published data
The published data on ChE contents in the CNG compared to normal levels are very sparse and contradictory. For example, information about Cl content in CNG was not found. Merely, one paper with results on Na level in normal thyroid and CNG was published in 1963 by Kamenev [52], but changes of this electrolyte level in goitrous thyroid were not shown. A relative good agreement there is only for I, since most of the published studies showed a significant decrease of I content in the CNG [53][54][55][56].
Information on the ChE contents in macro-or micro-follicular CNG, also about the association between ChE level and relative volume of colloid and cells in goitrous tissue, was not found.

Limitations
This study has some limitations. First, analytical techniques used in this study measure merely eight ChE (Br, Ca, Cl, I, K, Mg, Mn, and Na) mass fractions. Future studies should be aimed toward using other analytical methods which will elongate the list of ChE investigated in normal and goitrous thyroid. Second, the sample size of macro-or microfollicular CNG groups was relatively small and averted investigations of ChE contents in CNG group using differentials such as gender, stage of disease, and dietary habits of healthy persons and patients with CNG. Finally, the generalization of our outcomes may be bounded

Zaichick
to the Russian population. Despite these constraints, this study provides evidence on goiter-specific tissue Cl, I, and Na level alteration demonstrates associations between I and relative volume of colloid in CNG, and shows the necessity to continue ChE research of CNG of different histology.

CONCLUSION
In this work, ChE analysis was carried out in the tissue samples of normal and goitrous thyroid using INAA-SLR. It was shown that INAA-SLR is an adequate analytical tool for the non-destructive determination of Br, Ca, Cl, I, K, Mg, Mn, and Na content in the tissue samples of human thyroid in norm and pathology, including needle biopsy cores. It was perceived the considerable changes in ChE contents in the goitrous transformed tissue of thyroid, which depends on the histology of goiter. It was found that I predominately accumulates in colloid of CNG.