CC BY
38
PREVALENCE OF HASHIMOTO'S THYROIDITIS AMONG CHILDREN IN LVIV REGION DURING
YEARS 2000-2010
Olha Kasiyan,
Danylo Halytskyy Lviv National Medical University, Lviv, Ukraine Department of Hygiene and Preventive Toxicology;
Halyna Tkachenko,
Pomeranian University in Siupsk, Institute of Biology and Environmental Protection
Arciszewski Str. 22b, 76-200 Siupsk Jan Lukaszewicz Adam Mickiewicz University in Poznan Faculty of Geographical and Geological Sciences Institute of Physical Geography and Environmental Planning Department of Hydrology and Water Management Dzifgielowa Str. 27, 61-680 Poznan
Summary
The pathophysiology of autoimmune thyroid diseases during the past decades was described in details. Most of the factors involved in autoimmunity can be categorized into four groups: genetic, immune defects, hormonal and environmental factors (Hasham and Tomer, 2012). Genetic factors dominate, and influence with about 80% on the prevalence of immunity. Moreover, 20% of etiology is attributed to environmental factors (smoking, iodine intake, selenium deficiency, pollution, infectious conditions, physical and emotional stress) and physiological states (puberty, rapid growth, pregnancy, menopause, aging, female gender) (Beretic, 2011). The aim of our study was the assessment of Hashimoto's thyroiditis (HT) prevalence among children population in Lviv region (western Ukraine) during 20002010. HT prevalence among children population of different districts (reions) in Lviv region during 2000-2010 was done. Assessment of air quality, water and soil quality, and food quality according to the hygienic indicators (the number of samples that not match to standards, %) in districts of Lviv region in 2000, 2004 and 2010 years was also performed. Decrease of the HT prevalence from 4.7 to 2.7 per 10,000 children during 2000-2010 years was observed. A significant reduction in HT prevalence among children from large cities was noted. It was 7.1 per 10,000 individuals in 2000 and 2.1 per 10,000 individuals in 2010. There was a decline of HT prevalence from 3.2 to 2.4 per 10,000 individuals among children population from districts during 2000-2004, and its increase to 3.1 per 10,000 individuals from 2004 to 2010 years. In 2010, the rate of disease among children from towns was higher compared to the children from districts (3.1 and 2.1 per 10,000 individuals, respectively). The highest prevalence of HT among children was found in Brody, Turkivskyi, Zhovkivskyi and Horodok districts (9.4-27.2; 22.8-15.8, 2.4-5.3, 2.9-3.5 per 10,000 individuals, respectively), and in Drohobych, Stryi and Chervonograd (3.1-5.7, 3.4-6.8, 4.9-3.3 per 10,000 individuals, respectively). The increase of the prevalence of HT in 8 of the 20 districts and 2 of the 6 towns of Lviv region during 2000-2010 was observed. The increase of the HT prevalence in the Lviv region during 2000-2010 was mainly in the Brody, Peremyshliany and Turkivskyi districts. In these districts, high level of samples that not match to standards according to the air and food quality was observed. Moreover, the high relative risk of HT among children population in Lviv region during 2000-2010 years was also noted in Brody and Turkivskyi districts in 2000-2010 years, and Peremyshliany district during 2000-2004 years compared to Kamianka-Buzka as control district. Our study confirmed that mainly nutritive factors and environmental pollution by metals and chemicals are the main factors in the present-day spread of HT. Our results indicate the need to clarify the reasons for the increased HT prevalence among the population of Lviv region, the definition of the risk of thyroid pathology in each district of the region among the different age groups, which will propose measures to prevent further increase of HT incidence.
Keywords: autoimmune thyroid diseases, Hashimoto's thyroiditis, children, Lviv region, environmental pollution
INTRODUCTION
Autoimmune thyroid diseases (AITD), including Graves' disease (GD) and Hashimoto's thyroiditis (HT), are amongst the commonest autoimmune disorders, affecting approximately 5% of the population [13]. Enlargement of the thyroid gland, local tenderness and thyroid function disorder are common symptoms of thyroiditis. HT (chronic lymphocytic thyroiditis) is the most common form of thyroiditis in children. This disease is a frequent cause of acquired hypothyroidism. HT is characterized by infiltration of the thyroid gland by lymphocytes, gradual destruction of the gland and production of various thyroid autoantibodies, mainly
antimicrosomal and antithyroglobulin antibodies [22]. Patients with HT have positive antibodies to thyroglobulin and/or to thyroperoxidase in blood. Thyroid function could be normal or abnormal (overt hypothyroidism, subclinical hypothyroidism and hyperthyroidism). Abnormal ultrasound patterns may be present in patients with HT disease as diffuse hypoechogenicity and pseudonodules [18].
AITD is multifactorial with contributions from genetic and environmental factors [5]. It has been postulated that 79% of the susceptibility to develop AITD is attributed to genetic factors, while environmental factors contribute to 21% [20]. Genetic susceptibility in
combination with external factors are believed to initiate the autoimmune response to thyroid antigens in AITD. Indeed, there is solid epidemiological data to support a strong genetic influence on the etiology of AITD including family and twin studies [1]. Much information has been published about the genetic predisposition to AITD both in experimental animals and humans [5]. There is a shared genetic susceptibility to GD and HT. The putative GD and HT susceptibility genes include both immune modifying genes (e.g. HLA, CTLA-4) and thyroid specific genes (e.g. TSHR, Tg). Most likely, these loci interact and their interactions may influence disease phenotype and severity [1]. Among the major AITD susceptibility genes that have been identified and characterized is the HLA-DR gene locus, as well as non-MHC genes including the CTLA-4, CD40, PTPN22, thyroglobulin, and TSH receptor genes [23].
The exact nature of the role environmental factors play in AITD is still not well known, but the involvement of several factors such as iodine diet content, stress, drugs and infections has been reported [2, 20]. However, there is no clear evidence of causality and the mechanisms by which environmental factors trigger thyroid autoimmunity in genetically predisposed individuals remain not fully understood [20]. High iodine intake, selenium deficiency, pollutants such as tobacco smoke, infectious diseases such as chronic hepatitis C, and certain drugs are implicated in the development of autoimmune thyroiditis, primarily in genetically predisposed individuals [8]. The best-established environmental factor is excess dietary iodine. Increased iodine consumption is strongly implicated as a trigger for thyroiditis, but only in genetically susceptible individuals. However, excess iodine is not the only environmental agent implicated as a trigger leading to AITD [5]. Epidemiological data from countries with sufficient or high nutritional iodine intake clearly show that in comparison to countries with iodine deficiency there is an increase in the incidence of AITD and a change in the pattern of manifestation, e.g. in the proportion of autoimmune thyroiditis and hyperthyroidism due to Graves' disease [19].
In Ukraine, the following territories are biogeochemical iodine deficiency provinces: Zhitomir, Lviv, Ternopol, Volyn regions and other regions, and some regions of the Crimea as well [12]. It is necessary that food rations be supplemented with different iodine preparations with the view of preventing iodine deficiency [21]. Prophylaxis of endemic goiter has been doing in districts of Lviv region [12]. On the other hand, there is evidence to suggest that elevated levels of iodide in the diet are associated with AITD in susceptible individuals, and that HT is less common in susceptible individuals who live in regions with dietary iodine deficiency [13].
Incidence of AITD in Ukraine is currently higher than in past decades. In our previous study we
demonstrated increase of AITD prevalence in Lviv region during 2000-2010 was mainly by the adult population in the greater urban area, less - among adults of rural areas [15-17]. Increase in the AIT prevalence among adults identified in 16 districts and 3 towns of Lviv region [17]. The aim of our study was the assessment of HT prevalence among children population in Lviv region (western Ukraine) during 2000-2010.
MATERIALS AND METHODS For assessment of the HT prevalence among children population of different districts (reions) in Lviv region during 2000-2010, the analysis of archival data from Lviv Regional Endocrinology Hospital was done. The research study covered by 20 districts (reions) of Lviv region (Brody, Busk, Horodok, Drohobych, Zhydachivskyi, Zhovkivskyi, Zolochivskyi, Kamianka-Buzka, Mykolaiv, Mostyska, Peremyshliany, Pustomyty, Radekhiv, Sambir, Skole, Sokal, Starosambirskyi, Stryiskyi, Turkivskyi, and Yavorivskyi Districts), as well as six towns of Lviv region (Lviv, Boryslav, Drohobych, Stryi, Truskavets, Chervonograd). For assessment of the relative risk (RR) of HT among children population of Lviv region, retrospective study was used. Kamianka-Buzka district as control district for RR assessment in retrospective analysis was used. Assessment of air quality, water and soil quality, and food quality according to the hygienic indicators (the number of samples that not match to standards, %) in districts of Lviv region in 2000, 2004 and 2010 years was also done. The obtained results were analyzed statistically using the Statistica 10.0 software package (StatSoft, Poland). In order to find significant differences (significance level, p<0.05) between Hashimoto's thyroiditis prevalence in different districts of Lviv region, Kruskal-Wallis test was applied to the data [26].
RESULTS AND DISCUSSIONS Decrease of the HT prevalence from 4.7 to 2.7 per 10,000 children during 2000-2010 years was observed (Figs 1-3). A significant reduction in HT prevalence among children from large cities was noted. It was 7.1 per 10,000 individuals in 2000 and 2.1 per 10,000 individuals in 2010. There was a decline of HT prevalence from 3.2 to 2.4 per 10,000 individuals among children population from districts during 2000-2004, and its increase to 3.1 per 10,000 individuals from 2004 to 2010 years was observed. In 2010, the rate of disease among children from towns was higher compared to the children from districts (3.1 and 2.1 per 10,000 individuals, respectively).
The highest prevalence of HT among children was found in Brody, Turkivskyi, Zhovkivskyi and Horodok districts (9.4-27.2; 22.8-15.8, 2.4-5.3, 2.9-3.5 per 10,000 individuals, respectively), and in Drohobych, Stryi and Chervonograd (3.1-5.7, 3.4-6.8, 4.9-3.3 per 10,000 individuals, respectively). The increase in the prevalence of HT in 8 among 20 districts and 2 among 6 towns of Lviv region during 2000-2010 was observed
Fig. 1. Hashimoto's thyroiditis prevalence (per 10,000 individuals) among children population (A), as well as assessment of air quality (B), water and soil quality (C), and food quality (D) according to the
hygienic indicators (the number of samples that not match to standards, %) in districts (reions) of Lviv region in 2000.
Fig. 2. Hashimoto's thyroiditis prevalence (per 10,000 individuals) among children population (A), as well as assessment of air quality (B), water and soil quality (C), and food quality (D) according to the
hygienic indicators (the number of samples that not match to standards, %) in districts (reions) of Lviv region in 2004.
Fig. 3. Hashimoto's thyroiditis prevalence (per 10,000 individuals) among children population (A), as well as assessment of air quality (B), water and soil quality (C), and food quality (D) according to the hygienic indicators (the number of samples that not match to standards, %) in districts (reions) of Lviv region in 2010.
In our study, prevalence of Hashimoto's thyroiditis (per 10,000 individuals) among children population was the highest in Brody, Peremyshliany and Turkivskyi districts during 2000-2010. In these districts, high level of samples that not match to standards according to the air
quality was observed (Figs 1-3). Moreover, the number of samples that not match to standards according to the food quality in Brody district in 2000 and 2004 years was also noted (Figs 1 and 2).
The high relative risk (RR) of HT among children population in Lviv region during 2000-2010 years was noted in Brody district [4.48-26.87, p<0.05], in Turkivskyi district [10.85-21.28, p<0.001] in 2000-2010 years, and Peremyshliany district [4.50-14.20, p<0.05] during 2000-2004 years compared to Kamianka-Buzka as control district (Table 2).
Table 2. The relative risk of Hashimoto's thyroiditis among the children population in districts of Lviv region
during 2000-2010 years.
Districts of Lviv region The relative risk of Hashimoto's thyroiditis
2000 2002 2004 2006 2008 2010
Brody district 4.48* 1.28-15.7 9.92* 1.27-77.49 7.40* 0.93-59.2 8.83* 1.12-69.69 21.58* 2.91-159.8 26.87* 3.66-197.2
Peremyshliany district 4.50* 1.24-16.34 14.20* 1.82-110.9 9.85* 1.21-80.04 _ - -
Turkivskyi district 10.85* 3.33-35.36 - 18.70* 2.51-139.3 21.28* 2.87-157.9 19.53* 2.63-145.1 15.60* 2.08-117.2
significant difference is set (p<0.05) compared to control Kamianka-Buzka district.
In other districts of the Lviv region, the RR of HT among children population during 2000-2010 years was not changed (p>0.05). No differences in the RR of HT in children population from large industrial towns of Lviv region compared to the control city Boryslav (p>0.05) were found.
Increased RR and prevalence of Hashimoto's thyroiditis in Brody, Peremyshliany and Turkivskyi districts during 2000-2010 is connected with high level of environmental pollution. Indeed, such environmental pollutants as smoke, polychlorinated biphenyls, solvents and metals have been implicated in the autoimmune
process and inflammation. Environmental factors have not yet, however, been sufficiently investigated to clarify their roles in pathogenesis, and there is a need to assess their effects on development of the autoimmune process and the mechanisms of their interactions with susceptibility genes [8]. In our study, a significant impact of chemical pollution of water and soil on the RR of HT among residents of rural districts of the Lviv region was noted, while air pollution has impact on the RR of HT among urban residents. There is also increasing evidence that mainly nutritive factors and environmental pollution
by metals and chemicals are the main factors in the present-day spread of AITD [9].
There is a wide variety of synthetic chemicals that affect the thyroid gland or have the ability to promote immune dysfunction in the host. These chemicals are released into the environment by design, such as in pesticides, or as a by-product of industry. Candidate pollutants include polyaromatic hydrocarbons (PAH), polybrominated biphenols (PBBs), and polychlorinated biphenols (PCBs), among others. Infections are also reputed to trigger autoimmunity and may act alone or in concert with environmental chemicals. Infections are also reputed to trigger autoimmunity and may act alone or in concert with environmental chemicals [5].
The relative risk of Hashimoto's thyroiditis among the children population in districts of Lviv region can be connected with environment pollution. Indeed, Ukraine faces the same environmental problems. The rivers' contaminations are caused by two main types: on-going sources of pollution, and industrial accidents that cause ecological catastrophes. The sources of the on-going contaminations are factories near the river, or chemical fertilizers. Taking everything into account, the Carpathian rivers (e.g. Tisza, Laborec) often suffer from industrial contamination. There are many examples of recent chemical pollution (water and air), which are caused by industrial facilities from neighbouring countries [7]. According to Report of Helsinki Commission Baltic Marine Environment Protection Commission (2005) [10], approximately half of the diffuse nutrient pollution in Lviv region comes from livestock breeding, 30% from arable land and the rest from non-sewered sanitation systems. Furthermore, only 46% of the population is connected to sewerage systems and chlorinated pesticides are still used in the area. There are 16 municipal wastewater treatment plants in the Ukrainian part of the Bug River basin, the main river in Lviv region, each with a processing capacity of more than 150 m3/day. The plants were constructed during the 1970s and early 1980s, therefore the technology is outdated and the plants are in many cases overloaded. It is estimated that within 10 years nearly all of the existing plants will be unable to provide adequate treatment. Some 80% of the point source organic pollution load flowing into the Bug basin originates in Ukraine, particularly the Lviv region. Diffuse sources are the main contributors to nutrient pollution in the Bug River basin accounting for 84% of the total nitrogen pollution and 68% of the phosphorus pollution [10]. Absence of riparian areas and water protection zones also leads to negative consequences. The main polluters are housing and utilities companies. Particularly 'Lvivvodokanal' (Lviv city) is one among 100 biggest polluters in Ukraine. It contributed 66% of the sewage water and 78% of polluting substances disposed to the rivers of Lviv region in 2010 [6].
In Ukraine, the primary industries are manufacturing heavy machinery, chemicals, and timber. Ukrainian territory also are rich in different natural resources. There are many mineral deposits in western Ukraine [e.g. sulfur in the Yavoriv area, potassium salt in the Lviv and Ivano-Frankivsk regions (Stebnykiv, Dobrogostivsk, Boryslav, Kalush-Golyn, Trostyanets, Turo-Velytsk), table salt in Precarpathia and
Transcarpathia (Drogobych, Kalush, Dolyna, Solotvyno), zeolite tufa deposits in Transcarpathia] [7]. The area of western Ukraine is a transit route for the major east-west gas pipelines, which pass through Poland, Slovakia, and Hungary, and for the oil pipeline towards Slovakia. Refineries and processing plants, as well as oil and gas fields were installed in the Precarpathian region [7].
Ukraine has generally been an area of mild-to-moderate iodine deficiency [12]. The following territories in Ukraine are biogeochemical provinces with iodine deficiency: Zhitomir, Lviv, Ternopol, Volyn Oblast and other regions of Ukraine, and some regions of the Crimea as well. It is necessary that food rations be supplemented with different iodine preparations with the view of preventing iodine deficiency [12]. Preventative measures to eliminate iodine deficiency prior to 2001 had a negligible impact on the study population: only 5.4% of the population reported consuming iodinated preparations on an ongoing basis in the first round of screening. Over the years 2001-2003, an improvement in iodine nutrition of the population was noted; a statistically significant increase in urinary iodine excretion was observed and the screening questions on consumption of products containing stable iodine indicate a statistically significant increase in intake [24]. On the other hand, there is also evidence to suggest that elevated levels of iodide in the diet are associated with AITD in susceptible individuals, and that HT is less common in susceptible individuals who live in regions with dietary iodine deficiency [3, 4, 13]. Supplemental iodine significantly elevated incidence of iodine induced hyperthyroidism in endemic goiter area. Over the years 2001-2003, an improvement in iodine nutrition of the population was noted. There are epidemiologic studies in endemic goiter areas in Ukraine that report an increase in AITD, particularly HT, after the therapeutic administration of iodized salt [24].
In genetically predisposed individuals the iodine intake modulates autoimmune thyroid reactions. Especially with acute or chronic increase of iodine intake it leads to a significant increase in the incidence and intensity of autoimmune thyroid disease [19]. There are epidemiologic studies in endemic goiter areas in Ukraine that report an increase in AITD, particularly thyroiditis, after the therapeutic administration of iodized salt [24]. Lymphocytic infiltration of the thyroid is rarely found in patients from severe endemic goiter regions, yet there is a reversal of this observation after dietary iodine supplementation [11]. A threefold increase in the prevalence of autoimmune thyroiditis among schoolchildren was noted once iodine deficiency was eliminated in an area of endemic goiter in northwestern Greece [27]. Concomitantly, iodine restriction in many patients with primary hypothyroidism restored normal thyroid function [14, 25].
In summary, the highest prevalence of HT among children was found in Brody, Turkivskyi, Zhovkivskyi and Horodok districts (9.4-27.2; 22.8-15.8, 2.4-5.3, 2.93.5 per 10,000 individuals, respectively), and in Drohobych, Stryi and Chervonograd (3.1-5.7, 3.4-6.8, 4.9-3.3 per 10,000 individuals, respectively). The increase of the prevalence of HT in 8 of the 20 districts and 2 of the 6 towns of Lviv region during 2000-2010 was observed. The increase of the HT prevalence in the Lviv
region during 2000-2010 was mainly in the Brody, Peremyshliany and Turkivskyi districts. In these districts, high level of samples that not match to standards according to the air and food quality was observed. Moreover, the high relative risk of HT among children population in Lviv region during 2000-2010 years was also noted in Brody and Turkivskyi districts in 2000-2010 years, and Peremyshliany district during 2000-2004 years compared to Kamianka-Buzka as control district. Our results indicate the need to clarify the reasons for the increased HT prevalence among the population of Lviv region, the definition of the risk of thyroid pathology in each district of the region among the different age groups, which will propose measures to prevent further increase of HT incidence. Although approximately 70% of the risk for developing AIT is attributable to genetic background, environmental triggers are thought to play a role in the development of AITD in susceptible individuals in endemic goiter Lviv region.
References
1. Ban Y., Tomer Y. 2005. Susceptibility genes in thyroid autoimmunity. Clin. Dev. Immunol., 12(1): 4758.
2. Baretic M. 2011. 100 years of Hashimoto thyroiditis, still an intriguing disease. Acta Med. Croatica, 65(5): 453-457. [Article in Croatian, Abstract in English].
3. Brown R. 2013. Autoimmune thyroiditis in childhood. J. Clin. Res. Pediatr. Endocrinol., 5(1): 45-49.
4. Brown R.S. 2009. Autoimmune thyroid disease: unlocking a complex puzzle. Curr. Opin. Pediatr., 21(4): 523-528.
5. Burek C.L., Talor M.V. 2009. Environmental triggers of autoimmune thyroiditis. J. Autoimmun., 33(3-4):183-189.
6. Diachevska L. 2013. Geopolitical problems in nature management: case study for Ukrainian-Polish border region. P. 234-238. Access to the source: http://www. geokyiv.org/pdf/reports/41_Diachevska.pdf.
7. Dobak I. 2010. Environment and security in western Ukraine - short overview. Evfolyam 1. szam -2010. Marcius, pp. 110-118.
8. Duntas L.H. 2008. Environmental factors and autoimmune thyroiditis. Nat. Clin. Pract. Endocrinol. Metab., 4(8): 454-460.
9. Duntas L.H. 2011. Environmental factors and thyroid autoimmunity. Ann. Endocrinol. (Paris), 72(2): 108-113.
10. Evaluation of transboundary pollution loads. Report of Helsinki Commission Baltic Marine Environment Protection Commission. Helcom -environmental focal point information, 2005. 16 p.
11. Foley T.P.Jr. 1992. The relationship between autoimmune thyroid disease and iodine intake: a review. Endokrynol. Pol., 43, Suppl. 1: 53-69.
12. Gerasimov G. 2002. IDD in Eastern Europe and Central Asia. IDD Newsletter, Vol. 18(3), August.
13. Hasham A., Tomer Y. 2012. Genetic and epigenetic mechanisms in thyroid autoimmunity. Immunol. Res., 54(1-3): 204-213.
14. Kasagi K., Iwata M., Misaki T., Konishi J. 2003. Effect of iodine restriction on thyroid function in patients with primary hypothyroidism. Redox. Rep., 13: 561-567.
15. Kasian O., Manenko A., Tkachenko H., Kurhalyuk N. 2009. Retrospective analysis of morbidity for autoimmune thyroid disease in the Lviv region of Ukraine during 1999-2006. XLIII Symposium of the Polish Society for Histochemistry and Cytochemistry, Bydgoszcz, 21-23 September 2009. In: Medical and Biological Sciences, V. XXIII, Supl. 2: 49.
16. Kasiyan O., Manenko A., Kurhalyuk N., Tkachenko H., Kaminski P. 2010. Retrospective analysis of morbidity for autoimmune thyroid diseases in the Lviv region during 1999-2006. In book: "Globalizacja a problematyka ochrony srodowiska", red. T. Nocha, A. Wesolowskiej. Gdansk: Wydawnictwo Gdanskiej Wyzszej Szkoly Administracji, pp. 359-371.
17. Kasiyan O., Tkachenko H., Lukaszewicz J., Kurhaluk N. 2013. Assessment of autoimmune thyroiditis prevalence among Adults in Lviv Region during 20002010 years. Slupskie Prace Biologiczne, 10: 93-105.
18. Lorini R., Gastaldi R., Traggiai C., Perucchin P.P. 2003. Hashimoto's Thyroiditis. Pediatr. Endocrinol. Rev., 1(Suppl. 2): 205-211.
19. Schumm-Draeger P.M. 2004. Iodine and thyroid autoimmunity. Z. Arztl. Fortbild Qualitatssich., 98 Suppl 5: 73-76. [Article in German, Abstract in English].
20. Sgarbi J.A., Maciel R.M. 2009. Pathogenesis of autoimmune thyroid diseases. Arq. Bras. Endocrinol. Metabol., 53(1): 5-14. [Article in Portuguese, Abstract in English].
21. Shchelkunov L.F. 2000. Role of the iodine microelement in nutrition and prophylaxis of various diseases. Lik. Sprava, (6):81-83 [Article in Russian, Abstract in English].
22. Szymborska M., Staroszczyk B. 2000. Thyroiditis in children. Personal observations. Med. Wieku Rozwoj., 4(4): 383-391. [Article in Polish, Abstract in English].
23. Tomer Y., Huber A. 2009. The etiology of autoimmune thyroid disease: a story of genes and environment. J. Autoimmun., 32(3-4): 231-239.
24. Tronko M., Kravchenko V., Fink D., Hatch M., Turchin V., McConnell R., Shpak V., Brenner A., Robbins J., Lusanchuk I., Howe G. 2005. Iodine excretion in regions of Ukraine affected by the Chernobyl Accident: experience of the Ukrainian-American cohort study of thyroid cancer and other thyroid diseases. Thyroid, 15(11): 1291-1297.
25. Yoon S.J., Choi S.R., Kim D.M., Kim J.U., Kim K.W., Ahn C.W., Cha B.S., Lim S.K., Kim K.R., Lee H.C., Huh K.B. 2003. The effect of iodine restriction on thyroid function in patients with hypothyroidism due to Hashimoto's thyroiditis. Yonsei Med. J. 44: 227-235.
26. Zar J.H. 1999. Biostatistical Analysis, 4th ed., Prentice Hall Inc., New Jersey.
