Correlation of Dietary Zinc Intake and Serum Zinc with Thyroid Stimulating Hormone (TSH) and Free Thyroxine (FT4) Levels in Adult Hyperthyroid Patients

Background: Hyperthyroidism is a thyroid disorder caused by increased synthesis and secretion of thyroid hormone by the thyroid gland. Hyperthyroidism is characterized by low thyroid stimulating hormone (TSH) and high free thyroxine (FT4) levels. The prevalence of hyperthyroidism in Indonesia was 0.6% in female and 0.2% in male. Zinc acts as an enzyme cofactor that converts thyroxine into triiodothyronine and it affects TSH synthesis. Adequacy of dietary zinc intake and serum zinc levels in hyperthyroid patients indirectly affects TSH and FT4 hormones. Objective: to analyze the correlation of dietary zinc intake and serum zinc with TSH and FT4 levels in adult hyperthyroid patients. Materials and Methods: This cross sectional study sampling based on inclusion and exclusion criteria which obtained 50 adult hyperthyroid patients at the Clinic of the Magelang Health Research and Development Center. Data of TSH and FT4 levels were measured by the ELISA method, serum zinc was measured with the ICP-MS method, dietary zinc intake’s data was collected using the 24 hour dietary recall and it was analyzed based on the table of Indonesian food composition 2017. Statistical analysis used in this study were spearman correlation and multivariate linear regression with 95% confidence level. Results : Dietary zinc intake did not correlate with both TSH and FT4 levels (p>0.05). Serum zinc had no correlation with TSH level (p>0.05), while serum zinc had a positive correlation with FT4 level (r=0.327; β=0.054; p<0.05). Conclusion: Serum zinc concentrations in adult hyperthyroid patients must be maintained because abnormal zinc status in the body’s metabolism will affect FT4 level.


Introduction
Hyperthyroidism is collection of clinical symptoms caused by excess thyroid hormones. 1 The most common cause of hyperthyroidism is Grave's disease, an autoimmune disorder characterized by TSI (thyroid stimulating immunoglobulin) stimulate thyroid gland to increase thyroid hormones secretion. High levels of thyroxine (T4) as the main hormone of thyroid gland caused high levels of free thyroxine (FT4). TSI antibodies also bind TSH receptors in the thyroid gland that results in decreasing TSH levels. 1,2 The prevalence of hyperthyroidism was 1.2% in the United States population. 3 The National Baseline Health Research of Indonesia in 2007 found 12.8% male and 14.7% female with low TSH levels, 4 while based on the result of National Baseline Health Research 2013 there were 706,757 Indonesians aged ≥15 years who diagnosed with hyperthyroidism (0.4%). The prevalence of hyperthyroidism in Indonesia according to gender was 0.6% in women and 0.2% in men. 5 Central Java Province had 0.5% of hyperthyroid population, higher than the national prevalence of Indonesia. 6 Zinc is an essential mineral that needs in the body's metabolism and plays a key role in the formation of thyroid hormones. 7 Zinc is a cofactor of deiodinase enzymes which convert T4 to T3 and affect Thyrotropin releasing hormone (TRH) to stimulate TSH. 8,9 Zinc deficiency can caused by low dietary zinc intake and inadequate zinc absorption in the intestinal tract. 10 Study by Prasetyo et al. (2018) showed that 74,3% of Indonesian adult population tends to have low zinc intake. 11 Study in Pakistan showed that the mean level of serum zinc on hyperthyroid patients was lower than healthy subjects. 12 Another study found that serum zinc level in hyperthyroid patients was higher than hypothyroid patients and zinc supplementation in hyperthyroid patients can prevent thyroid function disorders. 8 Adequate zinc intake and serum zinc levels in hyperthyroid patients indirectly affect thyroid hormones. Therefore, this study aimed to analyze the correlation between dietary zinc intake and serum zinc with TSH and FT4 levels in adult hyperthyroid patients.

Materials and Methods
This study had a cross-sectional design study which was conducted at the Clinic of Magelang Health Research and Development Center, it is a referral clinic for the management and treatment of thyroid disorders, including hyperthyroidism. This research protocol was approved by the Ethics Committee of the Medical Faculty, Sebelas Maret University, Surakarta, Indonesia (No. 014 / UN27.06 / KEPK / EC / 2020) and conducted from February-July 2020. The population of this study was all adult hyperthyroid patients who have treatment at the Clinic of the Magelang Health Research and Development Center, there were 127 patients based on 2019 data. The sample was 50 patients which fulfilled inclusion and exclusion criteria. Formula of minimum sample size calculated based on limited population. 13 Inclusion criteria were hyperthyroid patients who are19-59 years old, TSH levels <0.3 mIU/ mL, and patients with or without stroma. Exclusion criteria included hyperthyroid patients who were taking zinc supplementation, pregnant, menstruation, and patients with chronic disease or malignancy. The independent variables were dietary zinc intake and serum levels. The dependent variables were TSH and FT4 levels. Phytate intake as a confounding variable. Dietary zinc and phytate intake was measured by the 24 hour dietary recall which interviewed 2 times on different days. 14 Serum zinc levels were obtained using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) method. 15 TSH and FT4 levels were measured by the ELISA method. 16 Data were collected after subjects agreed to participate in this study and filled out the informed consent. Blood sample of 5 cc was taken from vein in the arm's subject for serum zinc, TSH and FT4 levels analysis, then the researchers interviewed dietary intake and conducted anthropometric measurements on the subjects. Data was analyzed using the SPSS version 21 program. Normality of data was verified by Kolmogorov-Smirnov test. The correlation of dietary zinc intake and serum zinc with TSH and FT4 levels was analyzed using Rank Spearman correlation and multivariate linear regression. There were several limitations of this study, such as the sample size was small and high differences of the sample size in male and female subjects.

Results and Discussion Characteristics of Subjects
General characteristics of subjects in this study are presented in table 1. Subjects consisted of 41 female patients (82%) and 9 male patients (18%). The mean age of the subjects was 40.4 years old. Half of the subjects had normal nutritional status (52%). The mean score of BMI was 22.97 kg/m 2 .

TSH and FT4
The average level of TSH was 0.14 mIU/mL. Most of the subjects had low TSH levels (84%) and the rest had normal TSH levels (16%). The mean level of FT4 was 2.49 µg/dL. Subjects with high FT4 levels were 40%, normal FT4 levels were 54%, and low FT4 levels were 6%.  12 Serum zinc concentration has been widely used biochemical markers of zinc status. 14 Based on United Kingdom National Diet and Nutrition Survey, serum zinc concentration had significant positive correlation with dietary zinc intakes, which assessed by 7 day weighed food records. 20 Previous study in New Zealand showed that were significant negative correlations between serum zinc levels with phytate intake and phytate:zinc molar ratio. 21 Intakes of dietary zinc and phytate affect serum zinc concentration.

Correlation of dietary zinc intake and serum zinc with TSH and FT4
Zinc plays a key role in thyroid hormone metabolism. Zinc modulating transcription factors in thyroid hormone synthesis, regulating deiodinase enzyme activity, and synthesizes TRH and TSH. 9,22 TSH describes the action of thyroid hormones on thyrotropic cells and indicates the whole metabolic status of thyroid gland. A slight increase in thyroid hormones especially FT4, will decreased secretion of TSH about 10 times. 17 Thyroxine (T4) is the main hormone produced by thyroid gland, it is 93% of the total thyroid hormone. T4 released in a bound form with thyroxine-binding globulin (TBG), thyroxinebinding prealbumin (TBPA) and albumin. FT4 is a free form of thyroxine circulating in the bloodstream. Although just 0.03-0.05% FT4 in blood circulation, FT4 valid to describe hyperthyroidism state because it does not bind to protein. 23

*. Correlation is significant
The results of Rank-Spearman analysis between dietary zinc intake and serum zinc as independent variables with TSH and FT4 levels as dependent variables can be seen in Table 2. Serum zinc had no correlation with TSH levels (p>0.05) but it had positive correlation with FT4 ( r=0.33; p=0.02). Phytate intake as a confounding variable did not correlate with TSH and FT4 (p>0.05). Dietary zinc intake did not correlate with both TSH and FT4 (p>0.05). Zinc intake which is bound to phytate cannot be absorbed in the intestinal tract. 19 Inhibitory effect of phytate on zinc absorption depends on the dose of phytate against zinc. Therefore, phytate: zinc molar ratio was used to estimate the amount of absorbable zinc. 14 Dietary zinc intake in this study had medium absorption,  24 Based on animal study by Baltaci et al(2017), zinc supplementation can prevent abnormalities of thyroid hormone levels in rats with thyroid disorders. Zinc increases the production of thyroxine-binding protein that can change the levels of T4. 8 Zinc is an essential mineral needed in the function of I-5'deiodinase enzyme which converts T4 into the active form T3. Deiodinase enzymes act to remove iodine molecules from T4. 9 In conditions of zinc deficiency, activity of type I-5'deiodinase enzyme and conversion of T4 to T3 are limited which result in increases of T4 and FT4 hormones. 8 Abnormal serum zinc concentration will affect FT4 level in adult hyperthyroid patients, that it's important to maintain normal level of serum zinc. Hyperthyroid patients should choose animal foods as protein sources, such as liver, poultry, beef, fish, eggs, and milk as sources of zinc. 10 Animal foods do not contain phytates which resulted in better absorption of zinc from diet. 14

Conclusion
Based on our research in adult hperthyroid patients, serum zinc concentration had positive correlation with FT4 levels. Adult hyperthyroid patients who have a higher level of serum zinc than normal value had higher risk to have a high level of FT4. Adult hyperthyroid patients must be maintaining normal levels of serum zinc because it will affect FT4 level.

Ethical Approval:
Research involving human subjects complied with all relevant national regulations, institutional policies and is in accordance with the tenets of the Helsinki Declaration (as revised in 2013), and was stated as ethical conduct with the issuance of Ethical Clearance Number 014/UN27.06/KEPK/ EC/2020. Ethical clearence was approved by the Ethics Committee of Faculty of Medicine, Sebelas Maret University, Surakarta, Indonesia