Correlation of vitamin D level with thyroid status and TSH antibody titers in patients with Graves’ disease

Background. Various studies have shown the association of vitamin D deficiency with autoimmune diseases.  Studies  have  found  that  vitamin  D  levels  in  patients  with  autoimmune  thyroid  diseases  including Graves’ disease were lower than that in patients with non-autoimmune thyroid diseases such as toxic nodular goiter. Some studies have reported no such relationship between vitamin D level and autoimmune thyroid diseases. The purpose of the study:  to compare serum vitamin D level  in patients with Graves’ disease versus age and sex matched controls, to assess the correlation of vitamin D with thyroid status and thyrotropin receptor antibody titers. Materials and methods. 48 patients with Graves’ disease and 24 age and sex matched healthy individuals  were  recruited.  Hormonal  investigations  that  included  serum  thyroid  stimulating  hormone  (TSH), free thyroxine (fT4), free triiodothyronine (fT3), as well as calcium, 25-hydroxyvitamin D (25(OH)D), parathyroid hormone (PTH), thyroid stimulating hormone receptor antibody (TSH-Ab) were done for all subjects. Results. The patients with Graves’ disease had significantly  lower 25(OH)D  levels (16.3 ± 1.4 ng/ml) as compared  to control  subjects  (22.8 ± 1.6 ng/ml)  (p = 0.024). TSH  levels and TSH-Ab  titers differed significantly between vitamin D deficient Graves’ disease group (25(OH)D < 20 ng/ml) and vitamin D non deficient Graves’ disease group (25(OH)D ≥ 20 ng/ml). Thyroid volume did not differ significantly between these groups. Serum vitamin D level correlated significantly with TSH and TSH-Ab titers in patients with Graves’ disease. Conclusions. Serum vitamin D levels are significantly lower in patients with Graves’ disease. Significant correlation between vitamin D and TSH and TSH-Ab titers was found in these patients.


Introduction
Graves' disease is one of the common autoimmune thyroid disease encountered in routine clinical practice. In recent years, the extraskeletal effects of vitamin D have been extensively studied. Vitamin D deficiency is linked to a variety of autoimmune disorders, including autoimmune thyroid disease [1]. Several studies suggest that individuals with Graves' disease have lower vitamin D levels than the general population [2][3][4]; however, data on the relationship between the le-vels of vitamin D and clinical parameters in Graves' disease or therapeutic issues [5] are limited. A few studies have analyzed the association between serum vitamin D levels and Graves' disease, and available data remain inconclusive. Various studies have shown the association of vitamin D deficiency with autoimmune diseases. In past, studies have found that vitamin D levels in patients with autoimmune thyroid diseases including Graves' disease were lower than that in patients with non-autoimmune thyroid diseases [6,7].

Оригінальні дослідження /Original Researches/
To understand the role of vitamin D in Graves' disease, it is important to determine whether lower vitamin D le vels contribute to the development of Graves' disease. There are some animal data supporting this hypothesis. BALB/cJ mice given a vitamin D-deficient diet had lower preimmunization thyroxine levels and were more prone to developing persistent hyperthyroidism following immunization with the TSH receptor compared to mice fed regular chow. No differences in the TSH-Ab levels were observed, suggesting that vitamin D directly modulated thyroid function in this animal model [8]. The lack of a significant association between vitamin D and TSH receptor antibody would support the hypothesis that vitamin D deficiency might have a direct effect on the thyroid gland [9]. However, as all clinical studies that address this question have been crosssectional in design, it is impossible to conclude whether the vitamin D status is directly involved in the pathogenesis or a consequence of the disease.
Hence, the relationship between vitamin D and Graves' disease is still unresolved.
We purposed to compare serum vitamin D level in patients with Graves' disease versus age and sex matched controls and to find associations of vitamin D status and Graves' disease and ascertain any correlation of serum vitamin D level with various thyroid related parameters.

Materials and methods
A total of 48 patients (34 females and 14 males) with Graves' disease were recruited to this cross-sectional study. Graves' disease was diagnosed by clinical, hormonal evidence of hyperthyroidism (suppressed thyroid stimulating hormone (TSH) level with elevated free triiodothyronine (fT 3 ), and free thyroxine (fT 4 ) levels. Forty-four age and sex matched healthy individuals (21-55 years) meeting the exclusion criteria were taken as controls. To avoid influence of seasonal variation on vitamin D, we recruited controls within the same month (± 1 month) as that of cases.
Exclusion criteria included patients with liver, renal, metabolic bone, parathyroid disorders, any autoimmune diseases, history of intake of calcium and vitamin D supplements within preceding 3 months, current or recent use of steroids, antitubercular drugs, antiepileptics, ketoconazole, heparin, oral contraceptives and diuretics, pregnant, and lactating women. All study participants underwent a detailed history enquiry and clinical examination.
The thyroid gland correct volume was obtained using ellipsoid formula after measuring maximum the longitudinal (L), anteroposterior (AP), and transverse (T) axis of both lobes. Both lobe volumes were added to get the total volume of the gland. Written informed consent was taken from all study participants.
Descriptive statistical methods such as mean and standard deviation were applied to summarize continuous variables. Categorical data were summarized as percentages or proportions. Normality distribution of all parameters was checked using Shapiro-Wilk test. Categorical variables were compared using Chi-square test. Mann-Whitney U test and independent t tests were performed to compare means between two groups as required. Kruskal Wallis test was used for comparison of means between three or more groups. Pearson's correlation coefficient was used to analyze correlation between different parameters. A p-value of less than 0.05 was considered statistically significant.
This case-control study was performed with the Institutional Review Board protocol approval 25.02.2019 (number Table 1

Parameters
Graves' disease, n = 48 The study was conducted as part of a comprehensive scien tific research «Optimization of prevention, diagnosis and treatment of diabetes mellitus on the background of comorbid pathology, taking into account the effects of iodine and vitamin D deficiency» (Health Ministry of Ukraine registration 0120U000218).

Results
A total of 48 patients with Graves' disease and 24 age and sex matched controls were included in the study. The baseline characteristics of the study population are summarized in table 1. The mean age of patients with Graves' disease was 36.45 ± 8.60 years as compared to mean age of 33.75 ± 7.90 years among controls (p = 0.105). Graves' disease patients had significantly suppressed TSH levels, elevated fT 3 and ele vated fT 4 in comparison to controls (p < 0.001). The mean thyroid volume was significantly higher among Graves' disease patients (29.2 ± 8.3 cm 3 ) as compared to controls (12.5 ± 2.7 cm 3 ) (p < 0.05).
It was seen that mean serum 25(OH) D levels were significantly lower among Graves' disease patients (16.3 ± ± 1.4 ng/ml) versus controls (22.8 ± 1.6 ng/ml) (p = 0.024). The serum calcium and PTH levels did not vary significantly among Graves' disease and control groups.
The comparison of different parameters between groups with and without vitamin D deficiency are summarized in table 2. There wasn't significant difference among these two subgroups of Graves' disease patients with regard to mean serum TSH and serum TSH-Ab levels.

Discussion
We found that serum mean 25(OH) D levels were significantly lower in Graves' disease patients as compared to controls.
One of the first studies that reported widespread vitamin D deficiency among Graves' disease patients was reported by H. Yamashita et al. [10]. The mean level of vitamin D in men and women Graves' disease subjects were 41.3 ± 15.0 and 31.8 ± 13.3 nmol/l, respectively in their cohort. In 2013, T. Yasuda et al. reported significantly lower mean 25(OH)D level among new onset Graves' disease female patients versus controls and suggested toward association of vitamin D and Graves' disease [3]. The findings of low vitamin D status in Graves' disease of our study is in agreement to reports from previous studies [11][12][13]. In a recently published study from Sweden, authors have reaffirmed the finding of lower vitamin D levels in Graves' disease [9].
However, some studies have not reported similar finding of low vitamin D status among Graves' disease than control population [14]. In a meta-analysis to study the association of vitamin D and Graves' disease, authors concluded that the patients with Graves' disease were more likely to be deficient in vitamin D compared to the controls with a high heterogeneity [15].
In our study, we found statistical difference in terms of TSH and serum TSH antibody titers between groups with and without vitamin D deficiency. Only one previous study has looked into the difference of thyroid related parameters between two Graves' disease groups stratified on basis of mean serum vitamin D level (25(OH)D < 20 ng/ml group versus 25(OH)D > 20 ng/ml group) [14]. The authors noted that the two groups had no significant difference in terms of thyroid hormone status. They also reported that 100% of their Graves' disease patients were serum TSH antibody titers positive when 25(OH)D level < 20 ng/ml as compared to 37.5 % of patients were serum TSH antibody titers positive when 25(OH)D level > 20 ng/ml. Among our cohort, patients with lowest vitamin D levels (group with 25(OH)D < 20 ng/ml) had significantly the highest mean serum TSH antibody titers. Other authors [4] have noted lower mean 25(OH)D levels among serum TSH antibody titers positive Graves' disease patients in comparison to serum TSH antibody titers negative Graves' disease patients. Our results show that serum vitamin D level correlated with TSH level and serum TSH antibody titers in Graves' disease. Yasuda T. et al. [3] showed no association between serum 25(OH)D levels and thyroid function tests or serum TSH antibody titers. Recently published study [14]  Оригінальні дослідження /Original Researches/ also did not report any correlation of serum vitamin D level with thyroid hormones and thyroid auto-antibody titers. However, in contrast to these findings, two studies have showed significant inverse correlation between vitamin D levels and serum TSH antibody titers in Graves' disease [5,11]. From review of above findings, it seems that thyroid hormones are affected by vitamin D status in Graves' disease. Nevertheless, the relationship between vitamin D level and antibody titers need further clarification, though at present most of studies fail to report any significant correlation between these two parameters [14].
Our study has few limitations. We did not assess daily total dietary calcium, vitamin D, and phytate intake that may have affected vitamin D status. Serum 25(OH)D assay was done only once for each person. Being a cross-sectional study causality between vitamin D deficiency and risk of Graves' disease cannot be made. Our sample size was small, and hence, future large scale studies are essential to further clarify the findings.

Conclusions
Vitamin D levels are significantly lower in patients with Graves' disease. Correlation was observed between vitamin D and TSH level and serum TSH antibody titers in Graves' disease. Vitamin D deficiency is significantly associated with Graves' disease.

Conflicts of interests.
Authors declare the absence of any conflicts of interests and their own financial interest that might be construed to influence the results or interpretation of their manuscript.