Thyroid function and thyroglobulin level in iodine decient children of eastern Nepal

Objective: Both iodine deciency and excess can negatively impact thyroid function. The present study was conducted to assess iodine nutrition among children and thyroid function in iodine decient children. Results: A total of 1012 school aged children (6-14 years) from several schools of Udayapur district were enrolled initially for the assessment of urinary iodine concentration (UIC). Blood samples (n=83) were collected from a subgroup of children who had UIC<100 µg/L to measure serum thyroglobulin (Tg), thyroid stimulating hormone (TSH), free triiodothyronine (fT3) and free thyroxine (fT4). Serum UIC was measured by ammonium persulfate digestion method and Tg, TSH, fT4 and fT3 were measured by ELISA kits from Diametra Company. The prevalence of insucient UIC (UIC<100 µg/L) was 11.1% in school children’s of Udayapur district. The median UIC was 236 µg/L. The mean fT3, fT4 and TSH among children with insucient UIC were 2.55±0.43 pg/mL, 0.96±0.28 ng/dL and 3.60±1.44 mIU/L respectively. The Median Tg was 17.5 ng/mL. Overt hypothyroidism and subclinical hypothyroidism was seen in 6% and 3.6% cases with UIC<100 µg/L respectively.


Introduction
Iodine is an essential micronutrient required for production of thyroid hormones [1,2]. Iodine de ciency affects 2 billion people with 36.4% being school age children [3]. Iodine de ciency during pregnancy and childhood has severe consequences including developmental disorders, poor cognition and intellectual disability [4]. Supplementation of iodine in food or salt has led to signi cant reduction in iodine de ciency disorders worldwide [5].
Nepal has been continuously improving iodine nutrition after starting universal salt iodization program. A number of studies particularly in eastern Nepal show promising results in improvement of iodine de ciency as revealed by increase in median urinary iodine concentration (UIC) of school children [6][7][8].
However, there are also concerns about excess iodine intake in such population [9]. Iodine de ciency or excess both affects thyroid function, so it is important to maintain thyroid function in such population [1].
In this study we speci cally aimed to nd the recent iodine status and thyroid function in the children with inadequate iodine intake (median UIC < 100 µg/L and UIC:100-199 µg/L are considered as inadequate iodine intake and adequate iodine intake respectively as per WHO criteria) in Udayapur district.

Main Text
This was a community based cross-sectional study conducted in school children during September 2014 to August 2015 in Udayapur district of eastern Nepal. The study was conducted by the department of biochemistry in collaboration with department of pediatrics of B. P. Koirala Institute of Health Sciences (BPKIHS), Dharan, Nepal. A multistage random sampling technique was used to enroll school children aged 6 to 14 years in the study. Total of 1012 children from 4 VDCs (Beltar, Basaha, Rampur and Chaudandi) of Udayapur district participated in the study. In the rst stage, urine samples were collected from all participants and based on the UIC data, 83 blood samples out of 112 children who had UIC < 100 µg/L were collected to assess thyroid function and thyroglobulin level. A written informed consent was obtained from the guardian of children prior to participation in study. All the stake holders including participants were informed about the purpose of the study. All healthy school going children (aged 6-14 years of age) were included in the study. Children taking drugs that interfere in thyroid function, taking iodine supplement or with any severe illness were excluded. Anthropometric measurements (height and weight) were taken, and spot urine samples were collected from all participants. Urine samples were collected in a clean, tightly screw-capped plastic vial and transported to the biochemistry laboratory for analysis. Blood samples (3 ml) were collected in plain vial by venipuncture following the standard protocol. Clotted blood was centrifuged at 3000 rpm and serum was separated. Serum and urine samples were refrigerated at -20 ºC until analysis. Urinary iodine concentration was estimated by ammonium persulfate digestion microplate (APDM) method using Sandell-Kolthoff's reaction [10]. Serum free tri-iodothyronine (T3), free tetra-iodothyronine (T4), TSH and thyroglobulin (Tg) were measured by ELISA using commercial kits (Diametra Company).
The data was analyzed using SPSS version 20.0. The data are presented in form of mean ± SD, median with inter quartiles (IQR) and frequency (percentage). Chi-square test was applied to see statistical difference. Spearman's and Pearson's correlation analysis was used to nd relationship between quantitative variables. P value of ≤ 0.05 was considered statistically signi cant at 95% con dence interval.  Table 1 shows the classi cation of iodine nutrition status in the study participants according to modi ed WHO assessment criteria with adequate and more than adequate group merged as su cient category [3].
The overall prevalence of iodine de ciency based on individual UIC cutoffs in our study was 11.1% (i.e severe, moderate and mild de ciency).

ID = Iodine de ciency
The data is expressed as frequency (percentage). Chi-square test was used between gender and iodine status at 95% con dence interval.

UIC = Urinary iodine concentration
The data is expressed as frequency (percentage). Chi-square test was used between gender and thyroid function status at 95% con dence interval.
Iodine de ciency has remained as the most common cause of preventable brain damage in children worldwide [11]. Supplementation of iodine in salt through universal salt iodization has become effective in Nepal as shown by improving median UIC in the previous studies from eastern Nepal [6][7][8]. Median UIC indicates recent iodine intake and is used as marker of iodine status in the community settings. In the present study, median UIC was 236.0 µg/L which indicates su cient iodine nutrition among children of this district. Previous report showed median UIC of 268.02 µg/L among children of this district [7].
In this study 11.1% children had UIC < 100 µg/L which is considered as iodine de ciency. In the previous study, iodine de ciency in this district was 12.7% [7]. National surveys conducted in Nepal in the year 1998, 2005 and 2007 depicted iodine de ciency in 43.6%, 27.4% and 19.4% school age children respectively [12]. In another study in eastern regions, Dhankuta and Dharan, iodine de ciency was 26.6% and 15.6% respectively among school children [13,14]. Our ndings suggest improvement in iodine nutrition in this region over previous years. This reveals the effectiveness of universal salt iodization program in Nepal. The uctuation in diet across different seasons are also said to affect UIC of population leading to variation of UIC across various seasons in the same population [15].
Thyroglobulin, a thyroid-speci c protein and precursor in the synthesis of thyroid hormones, is also considered as a sensitive marker of the iodine status than UIC [16]. The median thyroglobulin level in the children with insu cient UIC was 17.5 ng/mL. Our previous study among pregnant women of eastern Nepal reported median Tg of 6.5 ng/mL [17]. Thyroglobulin increment in plasma is also seen with thyroid mass, in ammation and hyperactivity of TSH [18].
We observed normal thyroid function in majority of children with iodine de ciency. Out of 83 children with insu cient UIC, 6% (n = 5) had overt hypothyroidism and 3.6% (n = 3) had subclinical hypothyroidism.  [19,14,20]. Thyroid dysfunction, speci cally hypothyroidism was less common in this study than shown by our previous studies. This might be due to thyroid function assessment only in the subgroup with low UIC. In addition, there are other causative factors for thyroid disorders such as thyroid autoimmunity which might have been missed from this subgroup [17]. Thyroid function is said to be impaired in both the condition of iodine de ciency and excess [1].
We observed weak negative correlation of UIC with Tg level and thyroid hormones in children with insu cient UIC, and a positive correlation between thyroglobulin and fT3 (r = 0.273, p = 0.013). In iodine de cient cases, thyroglobulin tends to rise due to hyperactivity of thyroid gland and TSH however thyroid gland may be able to adapt to short term iodine de ciency maintaining normal thyroid hormones [1].
In summary, we found adequate iodine nutrition among the school children with small fraction having recent iodine de ciency, with the majority of children showing normal thyroid function.

Limitations
Page 6/9 The present study has several limitations. Individual UIC was used as a marker of iodine de ciency and thyroid function was not assessed in all the participants. Thyroglobulin was estimated in the children with low UIC and other causes of thyroid dysfunction were not investigated.

Declarations
Ethics approval and consent to participate The study was approved by Institutional Ethical Review Committee (IRC) of BPKIHS (Code No: IRC/422/014). Written consent was taken from children's parent before participation.

Consent for publication
Not necessary Availability of data and material Data available on reasonable request.

Competing interests
None Funding None Author's contribution SK1, SK2, BG, NB and ML designed the study. SK1 and ST collected the samples. SK1 analyzed the samples. SK2 performed data analysis and drafted the manuscript. SK1 helped in drafting manuscript. All authors read and approved the nal version of manuscript.