Outcome definition:
Normal thyroid function was considered as 0.3 mIU/L ≤ TSH ≤ 3.6 mIU/L. The diagnosis of overt and subclinical hypothyroidism respectively was done based on TSH levels higher than 10 and 3.6 mIU/L <TSH ≤ 10 mIU/L [18]. Normal T4 levels were considered between 4.5 and 12.0 μg/dL for normal participants. T4 value lower than 4.5 was one of the additional criteria’ for hypothyroidism patients [31]. The values higher than 40 and 100 IU/mL were considered positive for TPOAb and TGAb, respectively. Diagnosis criteria for HT included decreased T4 value along with an elevated TSH (Overt and subclinical hypothyroidism patients) and the presence of high serum TPOAb or TGAb concentrations. The patients having overt or subclinical hypothyroidism without positive TPOAb or TGAb were considered as having non-autoimmune hypothyroidism disease. Vitamin D levels lower than 8 ng/mL were considered as severe vitamin D deficiency, 9 – 15 ng/mL concentrations as mild vitamin D deficiency, higher than 16 to 20 ng/mL concentrations as vitamin D insufficiency and higher than 20 ng/mL concentrations as normal vitamin D level [32].
Laboratory measurements:
Blood samples were taken from all participants after at least 8 hours of fasting. T3, Free T4, TSH were measured by Cobas ECLIAs (Roche Diagnostics GmbH, Mannheim, Germany). Thyroid peroxidase antibody (TPOAb) were determined by chemiluminiscenta IMMULITE 2000 XPi (Siemens, Eschborn, Germany). Thyroid globulin antibody (TGAb) levels were analyzed by Enzyme-Linked Immunosorbent Assay (ELISA kit, Diesel). Vitamin D levels were measured by LIAISON vitamin D chemiluminescence immunoassay (DiaSorin, Saluggia, Italy).
Statistical methods:
In order to compare the quantitative continuous variables, ANOVA for parametric data and Man-u withney and Kruskal Wallis for non-parametric data were used. Chi-square test was used to compare discrete data among different groups. A p-value of less than 0.05 was considered statistically significant. SPSS v.19 was used for statistical analysis.
Findings
Totally 1138 individuals were studied. Demographic information and biochemical parameters of participants are presented in Table 1. Total vitamin D level of participants was 15.4(8.41-25.87). male participants had a higher level of vitamin D (p=0.001). There wasn’t any significant difference in the age of participants of Immune Hypothyroid, Non-Immune Hypothyroid and Control groups (p=0.630). Also, the distribution of male and female participants didn’t differ between there groups of the study (p=0.751).
Kruskal Wallis test revealed, there was a significant difference in Vitamin D level of study groups. As shown in figure2., Control subjects had significantly higher vitamin D level than both HT (p=0.001) and Non-autoimmune thyroid disease patients (p=0.001), but there wasn't any significant difference between vitamin D level of HT and Non-Immune Hypothyroid patients (p>0.05).
To investigate the relationship of the thyroid autoimmunity in HT patients and vitamin D, a chi-square test investigated the distribution of the vitamin D deficiency occurrence in TPOAb+ and TGAb+ patients. The results revealed a significantly higher rate of TPOAb+ in vitamin D deficient patients (p=0.030), however, there weren't any differences in the occurrence of TGAb+ in vitamin D deficient and sufficient patients (p=0.14).
The spearman test revealed that in HT patients there was a significant inverse correlation between the vitamin D and TGAb level (p=0.001, r=-0.261) and a direct correlation of vitamin D with TSH level (p=0.008, r=0.108). However, there wasn't any significant correlation between vitamin D and other paraclinical findings). Also, these correlations were not statistically significant in Non-Immune Hypothyroid and Control.