ASSOCIATION OF VITAMIN D DEFICIENCY WITH HYPOTHYROIDISM IN NEPALESE POPULATION: A PILOT STUDY

Mahesh Prakash Bhatta 1,2 , Bhupendra Raj Pandey 3 , Anupa Lamichhane 4 , Mahendra Prasad Bhatt 2,4 , Mukund Joshi 5 , Alneil M. Hamza 6 and Dipendra Raj Pandeya*. 1. Department of Clinical Laboratory Sciences, Pokhara University, Pokhara, Nepal. 2. LifeCare Diagnostics and Research Center Dhangadhi Pvt. Ltd, Dhangadhi, Nepal. 3. United Reference Laboratory Pvt. Ltd. Pokhara, Nepal. 4. Gandaki Medical College Teaching Hospital and Research Center Pvt. Ltd. Pokhara, Nepal. 5. Seti Zonal Hospital, Dhangadhi, Nepal. 6. Department of Clinical Laboratory Science, College of Applied Medical Sciences, Al Jouf University, Al Qurayyat, Saudi Arabia. 7. Department of Biochemistry, Nepalese Army Institute of Health Sciences, Kathmandu, Nepal ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

The thyroid hormones often referred to as the major metabolic hormones; affect virtually every cell in the body. Hypothyroidism is define as deficiency of thyroid function, reduced production of FT3 and FT4 that lead to hyper secretion of pituitary hormone TSH (Kirsten, 2000). Thyroid disorder is defined as the abnormal thyroid stimulating hormones (TSH) with normal or abnormal thyroid hormones, free tri-iodothyronine (fT3) and free tetraiodothyronine(fT4) (Smyth, 2009). Thyroid disease is most common in general population and its prevalence has risen markedly in the last few decades affecting even younger age groups in the form of congenital hypothyroidism (Unnikrishnan et al., 2011). Thyroid disease is most common in general population and its prevalence increases with age. Globally, 300 million people are suffering from thyroid disorder. These disorders are 8 times more common in female than male (Sawin et al., 1985;Smyth, 2009). In Nepal, thyroid disorder is one of the major health problems with an average prevalence of about 30% (Baral et al., 2002;Aryal et al., 2010;Chaudhari et al., 2012). Nepalese population is at higher risk for thyroid dysfunction due to geographical locations with high hilly areas with higher prevalence of iodine deficiency disorders (Baral et al., 1999; Baral et al., 2002;Gelal et al., 2009).
Vitamin D mediates its effect through binding to vitamin D receptor (VDR), and activation of VDR-responsive genes (Pike et al., 2010). Initially thought to be vitamin D just regulating calcium homeostasis, research has shown that this steroid hormone affects more than 36 cell types which possess VDR including thyroid gland; it also has a role in regulating cell proliferation and differentiation (Murray et al., 2012). Vitamin D exerts its metabolic effects on skeletal, cardiovascular and reproductive systems. So, a lower level of Vitamin D is likely to aggravate the systemic abnormalities associated with hypothyroidism (Wang et al., 2008;Chopra et al., 2011). Despite enough sunshine, this unexpected insufficiency of Vitamin D levels among Nepalese has become a matter of concern yet; no international health organization or governmental body has declared a health emergency to warn the public about the urgent need of achieving sufficient vitamin D blood levels (Naeem, 2010). It is still unclear if any association exists between hypothyroidism and Vitamin D insufficiency. The main aim of this study was to examine the relationship between hypothyroidism as well as autoimmune thyroid disorder with vitamin D deficiency and to justify the its association with serum calcium levels in a central Nepalese population.

Materials and Methods:-
This analytical study was conducted in the Department of Immune serology and Biochemistry of United Reference Laboratory Pvt. Ltd., Pokhara, Nepal during the period from October 2013 to June 2015. A total of two hundred subjects were included in this study after an informed consent. This study was approved by Research review committee (RRV).
Group I "control group": consisted of 100 apparently healthy individuals 42 Male (42%) and 58 Female (58%) of comparable age group (42.89±14.29). None of them had any history of chronic medical diseases, intake of dietary supplements or thyroid disorder related drugs. They were not on vitamin D supplements. All subjects were following the same protocol.

712
Group II "Study group": Formed by 100 patients, 20 Male (20%) and 80 Female (80%), their mean ages ± S.D was 42.81±14.57 year. They were diagnosed as hypothyroid patients if TSH level were higher than 4.20 µIU/ml with lower levels of FT3 than 3.1 pmol/L and FT4 than 12.0pmol/L. With no history of thyroxine and hypolipidemic drugs were included in the study. Patients with chronic renal failure, liver diseases, pregnancy and diabetes mellitus were excluded from the study.
Laboratory measurements:-Venous blood sample was collected under sterile conditions in two different gel tubes for each subject with and without use of tourniquet. Serum was separated by the use of centrifuge, checked for hemolysis and if hemolyzed then that serum was discarded. Thyroid function test (FT3, FT4, and TSH), vitamin D [25(OH)D] and thyroperoxidase Antibody(Anti-TPO) was tested by electrochemiluminescence method (ECLIA) in Cobas e411 (Roche diagnostics, Germany).Calcium level was determined in accent 200 (PZ Cormay S.A, Poland).

Statistical analysis:-
The study data was analyzed by using SPSS program to compute descriptive parameters including mean and frequencies, and inferential statistics was used including student's t test to test the significance of the differences between the mean values of two continuous variables and Chi-squire test (X 2 ) test the difference in proportions categorical variables between two groups. The level of confidence (P˂0.05) was considered as cutoff value for significance.

Results:-
Hypothyroid patients (study group) and normal individuals (Control group) were enrolled in this study. For each group 100 subject (20 male and 80 female) were selected. The mean age of the study subjects was 43 ±15 years (range 18-83) and 43 ±14(range 20-87) years for control group with no statistical significant difference, P= 0.933. FT3, FT4, TSH, Anti TPO, Vit. D and calcium were studied to assess the association of hypothyroidism and vitamin D level in hypothyroid patients.
[Table1] shows the statistics of biochemical parameters computed for study and control groups which point out that results were found to be: FT3     Table 4] show the correlation that was run to determine the relationship between FT3, FT4, TSH, Anti TPO and Vit. D within study group gender type. For male the result indicates there was a negative correlation with all thyroid functions parameter, which was not statistically significant as an example the correlation between Vit. D and TSH was depicted in [ Figure 2]. For female the result indicates there was only a positive correlation between (Vit. D level and FT4) which was statistically significant (r =-0.226, n = 80, p = 0.044). As example the correlation between (Vit D and TSH), (Vit D level and FT4) were depicted in [ Figure 3] and [ Figure 4] respectively.

Discussion:-
Vitamin D is known for its primary role in bone and mineral homeostasis, and it has been shown recently that its deficiency is associated with various diseases such as cardiovascular disease, cancer, infection, diabetes and other chronic diseases (Holick, 2004;Andress, 2006;Bikle, 2008). Interestingly, it has been shown recently that vitamin D has potent immunomodulatory effects and plays important roles in the pathogenesis of autoimmune diseases (Vondra et al., 2015). Few studies have been conducted in order to find any significant association between the levels of vitamin D and hypothyroidism and to determine whether vitamin D deficiency involves in the pathogenesis of hypothyroidism or rather a consequence of the disease and those that yielded conflicting results.
To our knowledge, there are some researchers examined the prevalence of Vitamin D deficiency in Nepalese populations but our study was one from few studies aimed to examine the association of Vitamin D level with hypothyroidism in Nepal mainly Mid western region. We therefore undertook this study to evaluate the levels of vitamin D among patients with hypothyroidism compared to healthy controls who did not complain from hypothyroidism or any thyroid diseases.
Low levels of 25(OH) Vitamin D is now a commonly accepted finding in central Nepalese population and overt deficiency has often been associated with a number of clinical disorders. We observed in this study that vitamin D does have a role to play in hypothyroidism, hypothyroid patients had significantly lower levels of serum 25(OH) Vitamin D as compared to controls (p <0.001). This outcome was supported by Tahir et al. (Tahir et al., 2016) studied that illustrated deficiency of serum Vit D levels were significantly associated with degree and severity of hypothyroidism, conducted in Indian population. In support to our study, Richards study also reported that low thyroid hormones lead to a lack of Vitamin D (Richards, 2008 In our study, it is found that female had low vitamin D and calcium level while had high Anti-TPO and TSH level than male, but were not significantly correlated. We observed a negative correlation of serum 25(OH) Vitamin D levels with TSH in both male and female hypothyroid patients on Pearson's correlation analysis (r= -0.41, P=0.072 and r=-0.219, P=0.051), suggesting an inter relationship that exists between vitamin D insufficiency and hypothyroidism. It also states a putative role of vitamin D as a potential modifiable risk factor for hypothyroidism. In order to function, vitamin D must bind to its receptor VDR which is found in several cell types including thyroid gland (Norman, 2008 One of the earlier studies reported low vitamin D level in both hypothyroid and hyper thyroid postmenopausal women (Clements et al., 2008). However, in the present study, positive correlation between FT4 and vitamin D3 was statistically significant in females probably due to low mean vitamin D values observed in females. Goswami et al. (Goswami et al., 1998) have also reported similar result in hyperthyroidism patients. According to this study significant number of patients (46%) with hyperthyroidism in India had fat malabsorption. Absorption of fat soluble 717 vitamins such as vitamin D is likely to be impeded in such a state. However, due to less sample size in male there was no significant correlation noticed.

Conclusion:-
Study indicated that patients with hypothyroidism suffered from hypovitaminosis D which encourages the supplementation of Vitamin D in hypothyroid patients and screening of Vitamin D levels in hypothyroid patients. Further, large scale prospective studies will be required to establish a cause and effect relation of vitamin D deficiency in pathogenesis of hypothyroidism.

Disclosure:-
The authors declared no conflicts of interest.