According to the World Health Organization (WHO), thyroid diseases rank second among endocrine disorders after diabetes mellitus. More than 665 million people in the world have endemic goiter or suffer from other thyroid pathologies. Women are affected by thyroid diseases almost ten times more often than men. At the same time, according to the same statistics, the increase in the number of thyroid diseases in the world is 5% per year [1]. It has been suggested that risk factors for the development of thyroid disorders may be numerous factors, including genetics, radiation, autoimmune diseases, as well as adverse environmental factors, such as an increase in the content of various chemicals in the environment [2].

Chemical elements (ChE) are among these various chemicals, because their levels in the environment have increased significantly over the past hundred years as a result of the industrial revolution and the tremendous technological changes that have taken place in metallurgy, chemical production, electronics, agriculture, food processing and storage, cosmetics, pharmaceuticals and medicine. In connection with these changes, the levels and ratio of ChE entering the human body from the outside have been significantly disturbed, compared with the conditions in which human societies have lived for many millennia.

More than 50 years ago, we formulated the postulate about the somatic ChE homeostasis, which is now generally recognized [3]. According to this postulate, under evolutionary environmental conditions, the mechanisms of homeostasis of organisms maintain the levels and ratios of ChE in tissues and organs within certain limits. If the content of ChE in the environment changes significantly, the mechanisms of somatic homeostasis may respond inadequately. Inadequate response of homeostasis mechanisms leads to changes in ChE levels in tissues and organs, which, in turn, can affect their function and lead to the development of pathological conditions. The correctness of this conclusion was illustrated by us earlier on the example of the study of the role of ChE in the normal and pathophysiology of the prostate [4-24]. It was shown, in particular, that a special role in the development of pathological transformations of the prostate is played by disturbances in the relationship between ChE in the tissue and gland secretion. Moreover, it was found that changes in the relationship between ChE can be used as highly informative markers of various prostate diseases, including malignant tumors [25-39]. These findings stimulated our investigations of ChE relationships in thyroid tissue in normal and pathological conditions.

There are many studies regarding ChE content in human thyroid, using chemical techniques and instrumental methods [40-51]. However, among the published data, no works on the relationship of ChE in the normal human thyroid were found.

This work had three aims. The primary purpose of this study was to determine reliable values for the bromine (Br), calcium (Ca), chlorin (Cl), iodine (I), potassium (K), magnesium (Mg), manganese (Mn), and sodium (Na) mass fractions in the normal thyroid of subjects ranging from children to elderly females using instrumental neutron activation analysis with high resolution spectrometry of short-lived radionuclides (INAA-SLR) and calculate individual values of I/Br, I/Ca, I/Cl, I/K, I/Mg, I/Mn, and I/Na. The second aim was to compare the Br, Ca, Cl, I, K, Mg, Mn, and Na mass fractions in thyroid gland obtained in the study with published data. The final aim was to estimate the inter-thyroidal correlations between ChE contents and between I/ChE content ratios in normal thyroid of females and changes of these parameters with age.

All studies were approved by the Ethical Committees of the Medical Radiological Research Centre, 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.

Samples of the human thyroid were obtained from randomly selected autopsy specimens of 33 females (European-Caucasian) aged 3.5 to 87 years. All the deceased were citizens of Obninsk and had undergone routine autopsy at the Forensic Medicine Department of City Hospital, Obninsk. The available clinical data were reviewed for each subject. None of the subjects had a history of an intersex condition, endocrine disorder, or other chronic disease that could affect the normal development of the thyroid. None of the subjects were receiving medications or used any supplements known to affect thyroid ChE contents. The typical causes of sudden death of most of these subjects included trauma or suicide and also acute illness (cardiac insufficiency, stroke, embolism of pulmonary artery, alcohol poisoning). All right lobes of thyroid glands were divided into two portions using a titanium scalpel [52]. One tissue portion was reviewed by an anatomical pathologist while the other was used for the ChE content determination. A histological examination was used to control the age norm conformity as well as the unavailability of micro adenomatosis and latent cancer.

After the samples intended for ChE analysis were weighed, they were transferred to -20°C and stored until the day of transportation in the Medical Radiological Research Center, Obninsk, where all samples were freeze-dried and homogenized [53]. To determine the contents of the ChE by comparison with a known standard, aliquots of commercial, chemically pure compounds were used [54]. Ten subsamples of the Certified Reference Material (CRM) IAEA H-4 (animal muscle) and IAEA HH-1 (human hair) were analyzed to estimate the precision and accuracy of results. The CRM IAEA H-4 and IAEA HH-1 subsamples were prepared in the same way as the samples of dry homogenized thyroid tissue.

The pounded sample weighing about 100 mg was used for ChE measurement by INAA-SLR. The samples for INAA-SLR were sealed separately in thin polyethylene films washed beforehand with acetone and rectified alcohol. The sealed samples were placed in labeled polyethylene ampoules. The content of Br, 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. The neutron flux in the channel was 1.7 × 1013n cm−2 s−1. Ampoules with thyroid tissue samples, interlaboratory-made standards, and certified reference material were put into polyethylene rabbits and then irradiated separately for 180 s. Copper foils were used to assess neutron flux. The measurement of each sample was made twice, 1 and 120 min after irradiation. The duration of the first and second measurements was 10 and 20 min, respectively. A coaxial 98-cm3 Ge (Li) detector and a spectrometric unit (NUC 8100), including a PC-coupled multichannel analyzer, were used for measurements. The spectrometric unit provided 2.9-keV resolution at the 60Co 1,332-keV line. Details of used nuclear reactions, radionuclides, and gamma-energies were presented in our earlier publications concerning the INAA-SLR of ChE contents in human prostate and scalp hair [55-58].

A dedicated computer program for INAA-SLR mode optimization was used [59]. All thyroid samples were prepared in duplicate, and mean values of ChE 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 was calculated for ChE contents and I/ ChE content ratios.  Pearson's correlation coefficient was used in Microsoft Office Excel to calculate the relationship "age – ChE mass fraction" and “age – I/ChE mass fraction", as well as to identify inter-thyroidal relationships between different ChE contents and between different ChE content ratios.

Table 1 depicts comparison of our data for seven ChE in ten sub-samples of CRM IAEA H-4 (animal muscle) and IAEA HH-1 (human hair) with the corresponding certified values of ChE 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, Ca, Cl, I, K, Mg, Mn, and Na mass fractions, as well as I/Br, I/Ca, I/Cl, I/K, I/Mg, I/Mn, and I/Na mass fraction ratios in normal thyroid of females.

The comparison of our results with published data for the Br, Ca, Cl, I, K, Mg, Mn, and Na contents in the human thyroid is shown in Table 3.

To estimate the effect of age on the ChE contents and I/ ChE content ratios Pearson's correlation coefficient was used (Table 4).

The data of inter-thyroidal correlation (values of r – Pearson's coefficient of correlation) including all ChE and I/ChE content ratios identified by us are presented in Tables 5 and 6, respectively.

Good agreement of the Br, Ca, Cl, I, K, Mg, Mn, and Na contents analyzed by INAA-SLR 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 TE contents and I/TE content ratios in the normal female thyroid presented in Tables 2–6.

Table 1: NAASLR data of Br, Ca, Cl, I, K, Mg, Mn, and Na contents in certified reference material IAEA H-4 (animal muscle) and IAEA HH-1 (human hair) compared to certified values (mg/kg, dry mass basis).

M-Arithmetical Mean, SD- Standard Deviation, a – Certified values, b – Information values.

The content of ChE 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, Ca, Cl, I, K, Mg, Mn, and Na mass fractions, as well as I/Br, I/Ca, I/Cl, I/K, I/Mg, I/Mn, and I/Na mass fraction ratios in normal thyroid of females (Table 2).

Table 2: Some statistical parameters of Br, Ca, Cl, I, K, Mg, Mn, and Na mass fraction (mg/kg, dry mass basis) as well as I/Br, I/Ca, I/Cl, I/K, I/Mg, I/Mn, and I/Na mass fraction ratios in normal thyroid of female (n=33).

Mean- Arithmetic Mean, SD- Standard Deviation, SEM- Standard Error of Mean, Min- Minimum Value, Max-Maximum value, P 0.025 – percentile with 0.025 level.

The values of M, SD, and SEM can be used to compare data for different groups of samples only under the condition of a normal distribution of the results of determining the content of ChE 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 Med, 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 ChE content in thyroid tissue. The obtained means for Br, Ca, Cl, I, K, Mg, Mn, and Na mass fraction, as shown in (Table 3).

Table 3: Median, minimum and maximum value of means Br, Ca, Cl, I, K, Mg, Mn, and Na contents in normal human thyroid according to data from the literature in comparison with our results (mg/kg, dry mass basis).

M-Arithmetic Mean, SD-Standard Deviation, (N)*- Number of All References, (N)**-Number of Samples.

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 [40-51]. A number of values for ChE mass fractions were not expressed on a dry mass basis by the authors of the cited references. However, we calculated these values using published data for water (75%) [60] and ash (4.16% on dry mass basis) [61] contents in thyroid of adults. No published data referring to I/Br, I/Ca, I/Cl, I/K, I/Mg, I/Mn, and I/Na mass fraction ratios in human thyroid was found.

With age, the Ca content, as well as I/Mg and I/Mn content ratios increase, while Cl content and I/Br content ratio decrease (Table 4). These characteristics, particularly I/Mg and I/Mn, can be used to estimate the "biological age" of the male thyroid gland.

Table 4: Correlations between age (years) and chemical element content (mg/kg, dry tissue), as well as between age and I/chemical element mass fraction ratios in the normal thyroid of females (r – coefficient of correlation).

Statistically significant values: a p≤0.05, b p≤0.01, c p≤0.001.

A significant direct correlation between the Br and K, Br and Mn, Cl and Mg, K and Mg, Na and Cl mass fractions, as well as an inverse correlation between Ca and Cl, and also Mn and Na mass fractions were seen in female thyroid (Table 5).

Table 5: Intercorrelations of the chemical element mass fractions in the normal thyroid of female (r – coefficient of correlation).

Statistically significant values: a p≤0.05, b p≤0.01, c p≤0.001.

Since no correlations were found between I and other ChE, it would appear that the content of Br, Ca, Cl, K, Mg, Mn, and Na in the thyroid gland is independent of I content. However, this is not quite so. If we bring the content of the studied ChE to the content of I (I/ ChE ratio), then there are close relationships between I/Br, I/Ca, I/Cl, I/K, I/Mg, I/Mn, and I/Na (Table 6).

Table 6: Intercorrelations of the I/chemical element mass fraction ratios in the normal thyroid of female (r – coefficient of correlation).

Statistically significant values: a p≤0.05, b p≤0.01, c p≤0.001.

From this it follows that, at least, the levels of all these ChE in the thyroid gland are interconnected and depend on the content of I in it.  Because I plays a decisive role in the function of the thyroid gland, the data obtained allow us to conclude that, along with I, such ChE as Br, Ca, Cl, K, Mg, Mn, and Na, if not directly, then indirectly, are involved in the process of thyroid hormone synthesis.

The INAA-SLR is a useful analytical tool for the non-destructive determination of ChE contents in the thyroid tissue samples. This method allows determine means for Br, Ca, Cl, I, K, Mg, Mn, and Na (eight ChE). Our data reveal that the Ca content, as well as I/Mg and I/Mn content ratios increase, while Cl content and I/Br content ratio decrease in the normal thyroid of female during a lifespan. Therefore, a goitrogenic and tumorogenic effect of excessive Ca level in the thyroid of old females and of disturbance in intrathyroidal I/Mg and I/Mn relationships with increasing age may be assumed. Furthermore, it was found that the levels of Br, Ca, Cl, K, Mg, Mn, and Na in the thyroid gland are interconnected and depend on the content of I in it. Because I plays a decisive role in the function of the thyroid gland, the data obtained allow us to conclude that, along with I, such ChE as Br, Ca, Cl, K, Mg, Mn, and Na, if not directly, then indirectly, are involved in the process of thyroid hormone synthesis.

Author is grateful to Dr. Yu. Choporov, Head of the Forensic Medicine Department of City Hospital, Obninsk, for supplying thyroid samples.

1. Wang H, Jiang Y, Song J, Liang H, Liu Y, Huang J, Tin P, Wu D, Zhang H, Liu, Zhon D, Wei W, Lei L, Peng J, Zhang J, et.al. The risk of perchlorate and iodine on the incidence of thyroid tumors and nodular goiter: a case-control study in southeastern China. Environ Health. 2022; 21: 4.
2. Tang Z, Zhang J, Zhou Q, Xu S, Cai Z, Jiang G, et.al. Thyroid cancer "epidemic": a socio-environmental health problem needs collaborative efforts. Environ Sci Technol. 2020; 54: 3725-3727.
3. Zaichick V. Medical elementology as a new scientific discipline. J Radioanal Nucl Chem. 2006: 269: 303-309.
4. Zaichick V. A systematic review of the mercury content of the normal human prostate gland. Archives of Urology 2020; 3: 35-45.
5. Zaichick V. A systematic review of the cobalt content of the normal human prostate gland. J Clin Res Oncol. 2020; 3: 1-8.
6. Zaichick V. A systematic review of the phosphorus content of the normal human prostate gland. Applied Medical Research 2020; 7: 1-7.
7. Zaichick V. A systematic review of the antimony content of the normal human prostate gland. Journal of Hematology and Oncology Research 2021; 4: 17-27.
8. Zaichick V. A systematic review of the chromium content of the normal human prostate gland. Innovare Journal of Medical Sciences 2021; 9: 1-6.
9. Zaichick V. A systematic review of the nickel content of the normal human prostate gland. Health Sciences 2020; 1: 1-6.
10. Zaichick V. A systematic review of the aluminum content of the normal human prostate gland. Advances in Hematology and Oncology Research 2021; 4(: 69-75.
11. Zaichick V. A systematic review of the lead content of the normal human prostate gland. J Cancer Oncol Res. 2021; 2: 1-8.
12. Zaichick V. A systematic review of the arsenic content of the normal human prostate gland. International Journal of Medicine Sciences 2021; 3: 1-6.
13. Zaichick V. A systematic review of the calcium content of the normal human prostate gland. Iberoamerican Journal of Medicine 2021; 3: 85-94
14. Zaichick V. A systematic review of the tin content of the normal human prostate gland. Journal of Cellular & Molecular Oncology 2021; 3: 1-7.
15. Zaichick V. Barium levels in the prostate of the normal human: a review. Universal Journal of Pharmaceutical Research 2021; 6: 52-60.
16. Zaichick V. A systematic review of the zinc content of the hyperplastic human prostate gland. Biomedical Research on Trace Elements 2020; 31: 98-116.
17. Zaichick V. Beryllium content of the normal human prostate gland: A systematic review. Acta Scientific Medical Sciences 2021; 5: 78-85.
18. Zaichick V. Boron level in the prostate of the normal human: A systematic review. J Nano Nano Sci Rese. 2021; 1: 1-9.
19. Zaichick V. Bismuth level in the prostate of normal human: A systematic review. Int J Biopro Biotechnol Advance 2021; 7: 264-273.
20. Zaichick V. A systematic review of the cadmium content of the normal human prostate gland. World Journal of Advanced Research and Reviews 2021; 10: 258-269.
21. Zaichick V. A systematic review of the strontium content of the normal human prostate gland. Journal of Medical Research and Health Sciences (JMRHS) 2021; 4: 1257-1269.
22. Zaichick V. Thallium content of the normal human prostate gland - A systematic review. Archives of Clinical Case Reports 2021; 2: 223-229.
23. Zaichick V. Vanadium content of the normal human prostate gland: A systematic review. Archives of Pharmacy Practice 2021; 12: 15-21.
24. Zaichick V, A systematic review of the zinc content of the normal human prostate gland. Biol Trace Elem Res.2021; 199: 3593-3607.
25. Zaichick V, Zaichick S. Ratios of selected chemical element contents in prostatic tissue as markers of malignancy. Hematol Med Oncol. 2016; 1: 1-8.
26. Zaichick V, Zaichick S. Ratios of Zn/trace element contents in prostate gland as carcinoma’s markers. Cancer Rep Rev. 2017; 1: 1-7.
27. Zaichick V, Zaichick S. Ratios of Mg/trace element contents in prostate gland as carcinoma’s markers. SAJ Canc Sci. 2017; 2: 102.
28. Zaichick V, Zaichick S. Ratios of calcium/trace elements as prostate cancer markers. J Oncol Res Ther. 2017; 2017: J116.
29. Zaichick V, Zaichick S. Ratios of cobalt/trace element contents in prostate gland as carcinoma’s markers. The International Journal of Cancer Epidemiology and Research 2017; 1: 21-27.
30. Zaichick V, Zaichick S. Ratios of cadmium/trace element contents in prostate gland as carcinoma’s markers. Canc Therapy & Oncol Int J. 2017; 4: 555626.
31. Zaichick V, Zaichick S. Ratios of selenium/trace element contents in prostate gland as carcinoma’s markers. J Tumor Med Prev. 2017; 1: 555556.
32. Zaichick V, Zaichick S. Ratios of rubidium/trace element contents in prostate gland as carcinoma’s markers. Can Res and Clin Oncology 2017; 1: 13-21.
33. Zaichick V, Zaichick S. Ratio of zinc to bromine, iron, rubidium, and strontium concentration in the prostatic fluid of patients with benign prostatic hyperplasia. Acta Scientific Medical Sciences 2019; 3: 49-56.
34. Zaichick V, Zaichick S. Ratio of zinc to bromine, iron, rubidium, and strontium concentration in expressed prostatic secretions as a source for biomarkers of prostatic cancer. American Journal of Research 2019; 5-6: 140-150.
35. Zaichick V, Zaichick S. Some trace element contents and ratios in prostatic fluids as ancillary diagnostic tools in distinguishing between the benign prostatic hyperplasia and chronic prostatitis. Archives of Urology 2019; 2: 12-20.
36. Zaichick V, Zaichick S. Some trace element contents and ratios in prostatic fluids as ancillary diagnostic tools in distinguishing between the chronic prostatitis and prostate cancer. Medical Research and Clinical Case Reports 2019; 3: 1-10.
37. Zaichick V, Zaichick S. Using prostatic fluid levels of zinc to strontium concentration ratio in non-invasive and highly accurate screening for prostate cancer. Acta Scientific Cancer Biology 2020; 4: 12-21.
38. Zaichick V, Zaichick S. Using prostatic fluid levels of zinc to iron concentration ratio in non-invasive and highly accurate screening for prostate cancer. SSRG International Journal of Medical Science 2019; 6: 24-31.
39. Zaichick V. Using prostatic fluid levels of rubidium and zinc concentration multiplication in non-invasive and highly accurate screening for prostate cancer. J Cancer Prev Curr Res. 2019; 10: 151-158.
40. Zhu H, Wang N, Zhang Y, Wu Q, Chen R, Gao J, Chang P, Liu Q, Fan T, Li J, Wang J, Wang J. Element contents in organs and tissues of Chinese adult men. Health Phys. 2010; 98: 61-73.
41. Salimi J, Moosavi K, Vatankhah S, Yaghoobi A. Investigation of heavy trace elements in neoplastic and non-neoplastic human thyroid tissue: A study by proton – induced X-ray emissions. Iran J Radiat Res. 2004; 1: 211-216.
42. Boulyga SF, Zhuk IV, Lomonosova EM, Kanash N, Bazhanova NN. Determination of microelements in thyroids of the inhabitants of Belarus by neutron activation analysis using the k0-method. J Radioanal Nucl Chem. 1997; 222: 11-14.
43. https://www.sciencedirect.com/science/article/abs/pii/S0168583X02012922
44. Woodard HQ, White DR. The composition of body tissues. Brit J Radiol. 1986; 708: 1209-1218.
45. Neimark II, Timoschnikov VM. Development of carcinoma of the thyroid gland in person residing in the focus of goiter endemic. Problemy Endocrinilogii 1978; 24: 28-32
46. Zabala J, Carrion N, Murillo M, Quintana M, Chirinos J, Seijas N, Duarte L, Brätter P. Determination of normal human intrathyroidal iodine in Caracas population. J Trace Elem Med Bio. 2009; 23: 9-14.
47. Forssen A. Inorganic elements in the human body. Annales Medicinae Experimentalis et Biologiae Fenniae. 1972; 50: 99-162
48. Kortev AI, Dontsov GI, Lyasheva AP. Bioelements and human pathology. Middle Ural book publishing house, Sverdlovsk, 1972, 302 p.
49. Soman SD, Joseph KT, Raut SJ, Mulay CD, Parameshwaran M, Panday VK. Studies of major and trace element content in human tissues. Health Phys. 1970; 19: 641-656.
50. Teraoka H. Distribution of 24 elements in the internal organs of normal males and the metallic workers in Japan. Arch Environ Health 1981; 36: 155-165.
51. Boulyga SF, Becker JS, Malenchenko AF, Dietze H-J. Application of ICP-MS for Multielement Analysis in Small Sample Amounts of Pathological Thyroid Tissue. Microchimica Acta 2000; 134(3-4): 215-222.
52. Zaichick V, Zaichick S. Instrumental effect on the contamination of biomedical samples in the course of sampling. The Journal of Analytical Chemistry 1996; 51: 1200-1205.
53. Zaichick V, Zaichick S. A search for losses of chemical elements during freeze-drying of biological materials. J Radioanal Nucl Chem. 1997; 218: 249-253.
54. Zaichick V. Applications of synthetic reference materials in the medical Radiological Research Centre. Fresenius J Anal Chem. 1995; 352: 219-223.
55. Zaichick S, Zaichick V. The effect of age and gender on 37 chemical element contents in scalp hair of healthy humans. Biol Trace Elem Res. 2010; 134: 41-54.
56. Zaichick S, Zaichick V. The scalp hair as a monitor for trace elements in biomonitoring of atmospheric pollution. IJEnvH 2011; 5: 106-124.
57. Zaichick S, Zaichick V. INAA application in the age dynamics assessment of Br, Ca, Cl, K, Mg, Mn, and Na content in the normal human prostate. J Radioanal Nucl Chem. 2011; 288: 197-202.
58. Zaichick V, Zaichick S. The effect of age on Br, Ca, Cl, K, Mg, Mn, and Na mass fraction in pediatric and young adult prostate glands investigated by neutron activation analysis. J Appl Radiat Isot. 2013; 82: 145-151.
59. Korelo AM, Zaichick V. Software to optimize the multielement INAA of medical and environmental samples. In: Activation Analysis in Environment Protection. Dubna, Russia; Joint Institute for Nuclear Research, 1993; 326-332.
60. Katoh Y, Sato T, Yamamoto Y. Determination of multielement concentrations in normal human organs from the Japanese. Biol Trace Elem Res. 2002; 90: 57-70.
61. Schroeder HA, Tipton IH, Nason AP. Trace metals in man: strontium and barium. J Chron Dis. 1972; 25: 491-517.