Journal of Pharmaceutical Research & Reports

Introduction

Thyroid benign nodules (TBNs) are universally encountered and frequently detected by palpation during a physical examination, or incidentally, during clinical imaging procedures. TBNs include non-neoplastic lesions, for example, colloid goiter and thyroiditis, as well as neoplastic lesions such as thyroid adenomas [1-3]. For over 20^th century, there was the dominant opinion that TBNs is the simple consequence of iodine deficiency. However, it was found that TBNs is a frequent disease even in those countries and regions where the population is never exposed to iodine shortage [4]. Moreover, it was shown that iodine excess has severe consequences on human health and associated with the presence of TBNs [5-8]. It was also demonstrated that besides the iodine deficiency and excess many other dietary, environmental, and occupational factors are associated with the TBNs incidence [9- 11]. Among these factors a disturbance of evolutionary stable input of many chemical elements (ChEs) in human body after industrial revolution plays a significant role in etiology of TBNs [12].

Besides iodine, many other ChEs have also essential physiological functions [13]. Essential or toxic (goitrogenic, mutagenic, carcinogenic) properties of ChEs depend on tissue-specific need or tolerance, respectively [13]. Excessive accumulation or an imbalance of the ChEs may disturb the cell functions and may result in cellular proliferation, 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 ChEs 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 many ChEs content with age in the thyroid of males and females were studied and age- and gender-dependence of some ChEs was observed [25- 41]. Furthermore, a significant difference between some ChEs contents in colloid goiter, thyroiditis, and thyroid adenoma in comparison with normal thyroid was demonstrated [42-46]. To date, the etiology and pathogenesis of TBNs must be considered as multifactorial. The present study was performed to find out differences in ChEs contents between the group of nodular tissues and tissue adjacent to nodules, as well as to clarify the role of some ChEs in the etiology of TBNs. Having this in mind, the aim of this exploratory study was to examine differences in the content of calcium (Ca), chlorine (Cl), iodine (I), potassium (K), magnesium (Mg), manganese (Mn), and sodium (Na) in nodular and adjacent to nodules tissues of thyroids with TBNs using a non-destructive instrumental neutron activation analysis with high resolution spectrometry of short-lived radionuclides (INAA-SLR), and to compare the levels of these ChEs in two groups (nodular and adjacent to nodules tissues) of the cohort of TBNs samples. Moreover, for understanding a possible role of ChEs in etiology and pathogenesis of TBNs results of the study were compared with previously obtained data for the same ChEs in “normal” thyroid tissue [42-46].

Material and Methods

All 79 patients suffered from TBNs (46 patients with colloid goiter, mean age M±SD was 48±12 years, range 30-64; 19 patients with thyroid adenoma, mean age M±SD was 41±11 years, range 22-55; and 14 patients with thyroiditis, mean age M±SD was 39±9 years, range 34-50) were hospitalized in the Head and Neck Department of the Medical Radiological Research Centre (MRRC), Obninsk. The group of patients with thyroiditis included 8 persons with Hashimoto’s thyroiditis and 6 persons with Riedel’s Struma. 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 ChEs contents. For all patients the diagnosis has been 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. 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 were divided into two portions using a titanium scalpel [47]. One was used for morphological study while the other was intended for ChEs analysis. After the samples intended for ChEs analysis were weighed, they were freeze-dried and homogenized [48]. The pounded samples weighing about 10 mg (for biopsy) and 100 mg (for resected materials) were used for ChEs measurement by INAA-SLR.

To determine contents of the ChE by comparison with a known standard, biological synthetic standards (BSS) prepared from phenol-formaldehyde resins were used [49]. In addition to BSS, aliquots of commercial, chemically pure compounds were also used as standards. Ten sub-samples of certified reference material (CRM) of the International Atomic Energy Agency (IAEA) IAEA H-4 (animal muscle) weighing about 100 mg were treated and analyzed in the same conditions as thyroid samples to estimate the precision and accuracy of results.

The content of Ca, Cl, I, K, Mg, Mn, and Na were determined by INAA-SLR using a horizontal channel equipped with the pneumatic rabbit system of the WWR-c research nuclear reactor (Branch of Karpov Institute, Obninsk). Details of used nuclear reactions, radionuclides, gamma-energies, spectrometric unit, sample preparation, and the quality control of results were presented in our earlier publications concerning the INAA-SLR of ChEs contents in human thyroid, scalp hair, and prostate [27,28,50-52].

A dedicated computer program for INAA-SLR mode optimization was used [53]. All thyroid samples for ChEs analysis were prepared in duplicate, and mean values of ChEs contents were used in final calculation. Using Microsoft Office Excel software, 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 ChEs contents in nodular and adjacent tissue of thyroids with TBNs. Data for “normal” thyroid were taken from our previous publications [42-46]. The difference in the results between three groups of samples (“normal”, “nodular”, and “adjacent”) was evaluated by the parametric Student’s t-test and non-parametric Wilcoxon-Mann-Whitney U-test.

Results

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 Ca, Cl, I, K, Mg, Mn, and Na mass fraction in “normal”, “nodular”, and “adjacent” groups of thyroid tissue samples.

Table 1: Some statistical parameters of Ca, Cl, I, K, Mg, Mn, and Na mass fraction (mg/kg, dry mass basis) in “normal” thyroid and thyroid benign nodules (nodular and adjacent tissue)

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.

The ratios of means and the comparison of mean values of Ca, Cl, I, K, Mg, Mn, and Na mass fractions in pairs of sample groups such as “normal” and “nodular”, “normal” and “adjacent”, and also “adjacent” and “nodular” are presented in Table 2, 3, and 4, respectively.

Table 2: Differences between mean values (M±SEM) of Ca, Cl, I, K, Mg, Mn, and Na mass fraction (mg/kg, dry mass basis) in “normal” thyroid (NT) and thyroid benign nodules (TBNs) (nodular tissue)

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

Table 3: Differences between mean values (M±SEM) of Ca, Cl, I, K, Mg, Mn, and Na mass fraction (mg/kg, dry mass basis) in “normal” thyroid (NT) and thyroid benign nodules (TBNs) (adjacent tissue)

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

Table 4: Differences between mean values (M±SEM) of Ca, Cl, I, K, Mg, Mn, and Na mass fraction (mg/kg, dry mass basis) in nodular and adjacent tissue of thyroid benign nodules (TBNs)

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

Discussion

As was shown before good agreement of the Ca, Cl, I, K, Mg, Mn, and Na contents in CRM IAEA H-4 samples analyzed by INAA- SLR with the certified data of this CRM indicates acceptable accuracy of the results obtained in the study of thyroid tissue samples presented in Tables 1-4[27,28,50-52].

The Cl, Mn, and Na contents in “nodular” tissue were higher, while Ca and I content was lower in comparison with contents of these ChEs in normal gland (Table 2). Significant differences between ChEs contents of “normal” thyroid and ChEs contents of thyroid tissue adjacent to nodules were found only for Cl and Na. Mass fractions of Cl and Na in “adjacent” group of samples were approximately 2.7 times and 60% higher, respectively, than in “normal” thyroid (Table 3). In a general sense Ca, Cl, K, Mg, Mn, and Na contents found in the “nodular” and “adjacent” groups of thyroid tissue samples were very similar (Table 4). However, level of I in “adjacent” group of samples was over 2 times higher than in nodular tissue (Table 4) and almost equals the normal value (Table 3).

Characteristically, elevated or reduced levels of ChEs 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 ChEs in affected thyroid tissues researchers try to determine the role of the deficiency or excess of each ChEs in the etiology and pathogenesis of thyroid diseases. In our opinion, abnormal levels of many ChEs in TBNs 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 ChEs level in pathologically altered tissue is the reason for alterations or vice versa. According to our opinion, investigation of ChEs contents in thyroid tissue adjacent to nodules and comparison obtained results with ChEs levels typical of “normal” thyroid gland may give additional useful information on the topic because this data show conditions of tissue in which TBNs were originated and developed.

Chlorine and sodium

Cl and Na are ubiquitous, extracellular electrolytes essential to more than one metabolic pathway. In the body, Cl and Na mostly present as sodium chloride. Therefore, as usual, there is a correlation between Na and Cl contents in tissues and fluids of human body. Because Cl is halogen like I, in the thyroid gland the biological behavior of chloride has to be similar to the biological behavior of iodide. The main source of natural Cl for human body is salt in food and chlorinated drinking water. Environment (air, water and food) polluted by artificial nonorganic Cl-contained compounds, for example such as sodium chlorate (NaClO₃), and organic Cl-contained compounds, for example such as polychlorinated biphenyls (PCBs) and dioxin, is other source. There is a clear association between using chlorinated drinking water, levels NaClO₃, PCBs and dioxin in environment and thyroid disorders, including cancer [54-58]. Thus, on the one hand, the accumulated data suggest that Cl level in thyroid tissue might be responsible for TBNs development. However, on the other hand, it is well known that Cl and Na mass fractions in human tissue samples depend mainly on the extracellular water volume [59]. Nodular and adjacent to nodules thyroid tissues can be more vascularized and can contain more relative volume of colloid than normal thyroid. Because blood and colloid are extracellular liquids, it is possible to speculate that it could be the reason for elevated levels of Cl and Na in TBNs and adjacent tissue. If that is the case, the equilibrium between Cl and Na increases has to be, however, in comparison with “normal” thyroid the change of Cl level in TBNs and adjacent tissue is significantly higher than change of Na level. Thus, it is possible to assume that an excessive accumulation of Cl in thyroid tissue is involved in TBNs etiology. Overall, the elevated levels of Cl in thyroid tissue could possibly be explored as risk factor of TBNs.

Iodine

To date, it was well established that I deficiency or excess has severe consequences on human health and associated with the presence of TBNs [5-8,54,60]. However, in present study neither reduced nor elevated levels of I in thyroid tissue adjacent to nodules in comparison with “normal” thyroid tissue were not found.

Compared to other soft tissues, the human thyroid gland has higher levels of I, because this element plays an important role in its normal functions, through the production of thyroid hormones (thyroxin and triiodothyronine) which are essential for cellular oxidation, growth, reproduction, and the activity of the central and autonomic nervous system. As was shown in present study, benign nodular transformation is probably accompanied by a partial loss of tissue-specific functional features, which leads to a modest reduction in I content associated with functional characteristics of the human thyroid tissue. Little reduced level of I content in nodular tissue could possibly be explored for differential diagnosis of TBNs and thyroid cancer, because, as was found in our ealier studies, thyroid malignant trasformation is accompanied by a drastically loss of I accumulation [18, 61-63].

This study has several limitations. Firstly, analytical techniques employed in this study measure only seven ChE (Ca, Cl, I, K, Mg, Mn, and Na) mass fractions. Future studies should be directed toward using other analytical methods which will extend the list of ChEs investigated in “normal” thyroid and in pathologically altered tissue. Secondly, the sample size of TBNs group was relatively small and prevented investigations of ChEs contents in this group using differentials like gender, histological types of TBNs, nodules functional activity, stage of disease, and dietary habits of patients with TBNs. Lastly, generalization of our results may be limited to Russian population. Despite these limitations, this study provides evidence on TBNs-specific tissue Cl, I, and Na level alteration and shows the necessity to continue ChEs research of thyroid benign nodules.

Conclusion

In this work, ChEs analysis was carried out in the tissue samples of TBNs using INAA-SLR. It was shown that INAA-SLR is an adequate analytical tool for the non-destructive determination of Ca, Cl, I, K, Mg, Mn, and Na content in the tissue samples of human thyroid in norm and pathology, including needle-biopsy specimens. It was observed that mass fractions of Cl and Na in “adjacent” group of samples were approximately 2.7 times and 60% higher, respectively, than in “normal” thyroid. In a general sense Ca, Cl, K, Mg, Mn, and Na contents found in the “nodular” and “adjacent” groups of thyroid tissue samples were very similar. However, level of I in “adjacent” group of samples was over 2 times higher than in nodular tissue and almost equals the normal value.

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, former Head of the Forensic Medicine Department of City Hospital, Obninsk, for supplying thyroid samples.

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