Differences of Relationships between Iodine and Trace Elements in Normal Thyroid and Thyroid Benign Nodules Revealed by X-Ray Fluorescent Analysis

Thyroid Benign Nodules (TBN) are the most common lesions of this endocrine gland. The etiology of TBN is not clear. The aim of this exploratory study was to examine differences in the content of bromine (Br), cooper (Cu), iron (Fe), iodine (I), rubidium (Rb), strontium (Sr), and zinc (Zn), as well as I/Br, I/Cu, I/Fe, I/Rb, I/Sr, and I/Zn content ratios in tissues of normal thyroid and TBN. Thyroid tissue levels of seven Trace Elements (TEs) were prospectively evaluated in 105 apparently healthy persons and in 79 patients with TBN. Measurements were performed using ¹⁰⁹Cd and ²⁴¹Am radionuclide-induced energy-dispersive X-ray fluorescent analysis. Tissue samples were divided into two portions. One was used for morphological study while the other was intended for TEs analysis. It was observed that in TBN the mass fraction of Br, Cu, and Fe were higher, whereas mass fractions of I as well as I/Br, I/Cu, I/Fe, and I/Zn mass fraction ratios were lower than in normal tissues of the thyroid. These changes can potentially be used as TBN markers. Furthermore, it was found that the levels of Br, Cu, Fe, Rb, Sr, and Zn contents in the normal and affected thyroid gland were interconnected and depend on the content of I in thyroid tissue. Because I plays a decisive role in the function of the thyroid gland, the data obtained allow us to conclude that, along with I, at least such TEs as Br, Cu, Fe, Rb, and Zn, if not directly, then indirectly, are involved in the process of thyroid hormone synthesis.

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 contents of other TEs. However, the situation changes significantly if, in studies of TEs relationships, not the absolute values of the TEs contents 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 bromine (Br), copper (Cu), iron (Fe), iodine (I), rubidium (Rb), strontium (Sr), and zinc (Zn) in TBN tissue using Energy Dispersive X-ray Analysis (EDXRF) and calculate individual values of I/TE ratios. TEs such as I, Cu, Fe and Zn play an exceptional role in the normal and pathological physiology of the body in general, and the thyroid gland in particular. EDXRF allows to simultaneously determine all these four TEs, as well as some others. Another aim was to compare the levels of these I/TE ratios in TBN with those in the normal thyroid. Finally, differences in intrathyroidal relationships of I/TE 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 contents 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 Kα-lines induced by radiation from radionuclide sources 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.

Detailed information about the EDXRF instruments with ¹⁰⁹Cd radionuclide source for determining the content of Br, Cu, Fe, Rb, Sr, Zn and with ²⁴¹Am radionuclide source for determining the content of I, as well as about methods of analysis and quality control of the results was presented in our earlier publications [21,23-26].

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 contents 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 contents ratios in normal thyroid and TBN.

Table 1 depicts comparison of our data for seven TEs in ten sub-samples of CRM IAEA H-4 (animal muscle) and 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 Br, Cu, Fe, I, Rb, Sr, and Zn mass fractions, as well as I/Br, I/Cu, I/Fe, I/Rb, I/Sr, and I/Zn mass fraction ratios in normal thyroid and TBN.

The comparison of our results with published data for the Br, Cu, Fe, I, Rb, Sr, and Zn contents in the human thyroid and TBN is shown in table 3.

Table 4 indicates the differences between mean values of Br, Cu, Fe, I, Rb, Sr, and Zn contents, as well as between mean values of I/Br, I/Cu, I/Fe, I/Rb, I/Sr, 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 and between I/TEs ratios identified by us in normal thyroid and TBN are presented in table 5.

Precision and accuracy of results

X-ray Fluorescence analysis (XRF) has many implementation options. In addition to EDXRF, there is a variant of Wave Dispersion (WDXRF), Total Reflection (TXRF), while the radiation of radionuclides, an X-ray tube, and even a synchrotron can be used to excite the characteristic X-ray radiation of atoms. The WDXRF in multi-elemental analysis takes much more time than the EDXRF. The variant of TXRF is a destructive method, i.e. requires acid digestion of the sample. We chose the cheapest version of the EDXRF device, which allowed us to quickly determine the contents of all the TEs under study without destroying the sample investigated.

Good agreement of the Br, Cu, Fe, I, Rb, Sr, and Zn contents analyzed by EDXRF with the certified data of CRMs IAEA H-4 and IAEA HH-1 (Table 1) indicates an acceptable accuracy of the results obtained in the study for TEs contents and I/TEs content ratios in the normal thyroid and TBN presented in tables 2-5.

The contents of TEs was determined in all or most of the examined samples, which made it possible to calculate the main statistical parameters: the mean value of the Mass fraction (M), Standard Deviation (SD), Standard Error of the Mean (SEM), Minimum (Min), Maximum (Max), Median (Med), and percentiles with levels of 0.025 (p = 0.025) and 0.975 (p = 0.975), of the Br, Cu, Fe, I, Rb, Sr, and Zn mass fractions, as well as I/Br, I/Cu, I/Fe, I/Rb, I/Sr, and I/Zn mass fraction ratios in normal thyroid and TBN (Table 2). The values ​​of M, SD, and SEM can be used to compare data for normal thyroid and TBN only under the condition of a normal distribution of the results of determining the content of TEs in the samples under study. Statistically reliable identification of the law of distribution of results requires large sample sizes, usually several hundred samples, and therefore is rarely used in biomedical research. In the conducted study, we could not prove or disprove the “normality” of the distribution of the results obtained due to the insufficient number of samples studied. Therefore, in addition to the M, SD, and SEM values, such statistical characteristics as Median, range (Min-Max) and percentiles p = 0.025 and p = 0.975 were calculated, which are valid for any law of distribution of the results of TEs contents in normal and pathological thyroid tissue.

Comparison with published data

The obtained means for Br, Cu, Fe, Rb, Sr, and Zn mass fraction, as shown in table 3, agree well with the medians of mean values cited by other researches for the human thyroid, including samples received from persons who died from different non-thyroid diseases [51-58]. Our means for Br, Cu, Fe, Rb, Sr, and Zn mass fraction in TBN (Table 3) are also within the range of published mean values for these TEs and agree well with their medians [59-66]. Some values for means of TEs mass fractions reported were not expressed on a dry mass basis. Because of this we recalculated these values using published data for water (75%) [67] and ash (4.16% on dry mass basis) [68] contents in thyroid of adults.

No published data referring I/Br, I/Cu, I/Fe, I/Rb, I/Sr, I/Zn in the normal thyroid gland and TBN were found.

The results shown in table 3 for the normal thyroid also includes samples from patients who died from various non-endocrine diseases. In our previous study, it was shown that some non-endocrine diseases can affect the contents of TEs in the thyroid gland [24]. Moreover, in many studies, "normal" thyroid refers to visually unaffected tissue adjacent to benign or malignant thyroid nodules. However, it was previously found that the tissue adjacent to benign or malignant thyroid nodules is not identical in its elemental composition to healthy thyroid tissue [69-74].

The range of means of Br, Cu, Fe, I, Rb, Sr, and Zn reported in the literature for normal thyroid and TBN vary widely (Table 3). This can be explained by the dependence of the TEs content on many factors, including the “normality” of the thyroid samples (see above), the region of the thyroid gland from which the sample was taken, age, gender, ethnicity, gland mass, and goiter stage. Not all these factors were strictly controlled in the cited studies. However, in our opinion, the main reasons for the variability in published data may be related to the accuracy of analytical methods, sample preparation methods, and the impossibility of taking homogeneous samples from affected tissues. It was insufficient quality control of results in these studies. In many scientific investigations, tissue samples were incinerated or dried at high temperature for many hours. In other cases, thyroid samples were treated with solvents (distilled water, ethanol, formalin, etc.). There is evidence that during ashing, drying and digestion at high temperature, significant amounts of some TEs are lost as a result of such processing. This applies not only to such volatile halogens as Br and I, but also to other TEs studied in the present work [75-77].

Differences between the normal thyroid and TBN in the contents of TEs and I/TEs relationships

From table 4, it is observed that in TBN the mass fraction of Br, Cu, and Fe were 29.6, 2.41, and 1.55 times higher, respectively, whereas mass fractions of I was 32% lower than in normal tissues of the thyroid. Since the changes in the content of Br, Cu, and Fe, on the one hand, and I, on the other hand, in TBN were in different directions, the ratios of I/Br, I/Cu, and I/Fe in TBN also differed significantly from the norm (Table 4). Moreover, the I/Zn ratio in TBN was significantly below (36%) the normal level. This confirmed that the I/TEs ratios are more sensitive parameters than the absolute values of the TEs contents in thyroid tissue.

Generally, elevated or decreased levels of TEs observed in TBN are discussed in terms of their potential role in the pathogenesis of TBN. In other words, researchers are trying to determine the role of deficiency or excess of each TEs in the occurrence of TBN by the low or high levels of TEs in TBN tissues. In our opinion, the abnormal levels of many TEs in TBN could be both a cause and a consequence of thyroid transformation. Thus, based on the results of such studies, it is not possible to decide whether the measured decrease or increase in the levels of TEs in pathologically altered tissue is the cause or consequence of the disease.

Relationships between trace elements in normal thyroid and TBN

A significant direct correlation between the mass fractions of Cu and Fe, as well as an inverse correlation between the mass fractions of I and Fe, was observed in the normal thyroid gland (Table 5). The absence of correlations between I and other TEs in the normal thyroid gland, with the exception of an inverse relationship between I and Fe, suggested that the content of Br, Cu, Rb, Sr, and Zn in the thyroid gland does not depend on the content of iodine. However, this is not quite true. When the contents of the studied TEs was reduced to the content of I (I/TEs ratios), it turned out that there are several significant direct correlations between I/Br, I/Cu, I/Fe, I/Rb, I/Sr, and I/Zn (Table 5).

In TBN, there were no correlations between Cu and Fe and between I and Fe, as well as an inverse correlation between the mass fractions of I and Fe, but new direct relationships between Br and Zn and between Cu and Sr appeared. As for the I/TEs ratios in TBN, all studied ratios (I/Br, I/Cu, I/Fe, I/Rb, I/Sr and I/Zn) were directly correlated with each other, except for I/Br and I/Sr (Table 5). It followed that, at least, the levels of Br, Cu, Fe, Rb, and Zn in the normal thyroid gland and TBN are interrelated and depend on the content of I in it and that along with I these TEs participate, if not directly, then indirectly, in the process of synthesis of thyroid hormones.

The ¹⁰⁹Cd and ²⁴¹Am radionuclide-induced EDXRF is a useful analytical tool for the non-destructive determination of TEs contents in the normal and affected thyroid tissue samples. This method allows determine means for Br, Cu, Fe, I, Rb, Sr, and Zn (seven TEs).

Our data reveal that in TBN the mass fraction of Br, Cu, and Fe were higher, whereas mass fractions of I as well as I/Br, I/Cu, I/Fe, and I/Zn mass fraction ratios were lower than in normal tissues of the thyroid. These changes can potentially be used as TBN markers. Furthermore, it was found that the levels of Br, Cu, Fe, Rb, Sr, and Zn contents in the normal and affected thyroid gland were interconnected and depend on the content of I in thyroid tissue. Because I plays a decisive role in the function of the thyroid gland, the data obtained allow us to conclude that, along with I, at least such TEs as Br, Cu, Fe, Rb, and Zn, if not directly, then indirectly, are involved in the process of thyroid hormone synthesis. It follows that for the normal functioning of the thyroid gland, it is necessary to maintain an adequate concentration of I in its tissue, balanced with the levels of other TEs.

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

Funding

There were no any sources of funding that have supported this work.

Conflict of Interest

The author has not declared any conflict of interests.

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