Thyroid Functions in Bamboo-Shoots Consuming Children of Tripura, North-east India

Information on thyroid functions in populations consuming potent antithyroidal bamboo-shoots (BS) was found scanty. Therefore, to assess thyroid function in BS consuming children was found relevant. Methods This cross-sectional study included 127 children from 3 villages. Thyroid volume (TV), free thyroxine (FT 4 ), free triiodothyronine (FT 3 ), thyroid stimulating hormone (TSH), thyroglobulin antibody (Tg-Ab), thyroid peroxidase antibody (TPO-Ab), urinary iodine (UI), urinary thiocyanate (USCN), salt iodine (SI), water iodine (WI), and BS consumption pattern were assessed. Results There were positive correlations between TV and FT 4 (r=0.2466, p=0.005), UI and TSH (r=0.2633, p=0.003), TSH and FT 4 (r=0.2135, p=0.016), TSH and FT 3 (r=0.1898, p=0.033), USCN and FT 4 (r=0.2477, p=0.005), Tg-Ab and TPO-Ab (r=0.3768, p=<0.001), and negative correlations between Tg-Ab and TSH (r=-0.2024, p=0.023), Tg-Ab and FT 4 (r=-0.1869, p=0.035). In boys, USCN had a positive correlation with TPO-Ab (r=0.3069, p=0.018). The village having highest levels of BS consumption (p=0.037) and median UI showed higher TSH levels (p=0.037) and higher FT 3 level (p=0.001) compared to the village of lowest BS consumption. sulphate transmittance urinary iodine interpolation classies hypothyroidism into subclinical (high TSH and normal FT 4 ), primary or overt (high TSH and low FT 4 ) and secondary or central (low TSH and low FT 4 ), and hyperthyroidism into subclinical (low TSH and normal FT 4 ), primary (low TSH and high FT 4 ) and secondary or TSH-mediated (high TSH and high FT 4 ) [38, 39]. Present study’s thyroid dysfunction prevalence (1.6% subclinical hypothyroidism and 4.7% subclinical hyperthyroidism) was lower than that of the area with adequate iodine [33]. Positive associations of TSH with FT 4 and FT 3 as observed suggest that levels of FT 4 and FT 3 were below their set points which led to compensatory increase in TSH that subsequently raised the levels of FT 4 and FT 3 in Thyroglobulin Is a Sensitive Measure of Both Decient and Excess Iodine Intakes in Children and Indicates No Adverse Effects on Thyroid Function in the UIC Range of 100–299 µg/L: UNICEF/ICCIDD


Introduction
Thyroid gland synthesizes thyroid hormones (TH) -thyroxine (T 4 ) and triiodothyronine (T 3 ) which play pivotal roles in growth, development and functions of body organs. For the synthesis of TH, iodine is essential in trace amount [1,2]. However, about 80% of T 3 is formed from extrathyroidal deiodination of T 4 [1]. As FT 4 and FT 3 (unbound forms) are accessible to peripheral tissues and regulate biological activities of T 4 and T 3 respectively, FT 4 and FT 3 are considered clinically most relevant for evaluating thyroid disorders [3]. Circulating levels of T 4 , T 3 and TSH remain relatively constant and re ect their 'set points' of hypothalamic-pituitary-thyroid (HPT) axis [4]. For each hormone, every individual has a HPT axis set point showing circadian rhythm determined by genetic makeup [5] and in uenced by factors like age [6], iodine intake [7], seasons [8], smoking [9], antithyroid substances and nutrient de ciencies [1]. Thyroid stimulating hormone stimulates TH synthesis and release, increasing Na + /I − symporter (NIS), thyroglobulin and thyroid peroxidase synthesis, iodine organi cation, and thyroglobulin turnover [10].
Natural sources of iodine are iodides of food and water [1]. An individual having a UI of 100-199 µg/L is considered to have adequate iodine nutrition, indicating an iodine intake of 150-<300 µg/day [11]. Individuals consuming foodstuffs de cient in iodine and/or rich in goitrogens may face iodine de ciency (ID) [1,2,12]. In ID, circulating levels of T 4 and T 3 remain generally lower and higher respectively than in iodine-su cient populations. Pituitary gland generally discharges TSH in response to circulating T 4 levels, and circulating TSH rises and falls when circulating T 4 levels remain low and high respectively. Iodine de ciency reduces T 4 and raises TSH in circulation, so circulating TSH remains generally higher in iodine-de cient populations than in iodine-su cient ones, and is extensively used as an indicator for both hypothyroidism and hyperthyroidism [11].
Prolonged TSH action forms goiter causing hypertrophy and hyperplasia of thyroid gland. On long standing, initial diffuse goiter becomes nodular and toxic with the appearance of hyperactive autonomous nodules producing excessive T 4 independent of TSH [1]. In addition to goiter and hypothyroidism, ID may also (a) result in mental retardation, deaf-mutism, squint, spastic diplegia, impaired learning capacity, and cretinism, (b) affect reproductive function leading to increased incidence of stillbirths, spontaneous abortions, congenital anomalies, low birth weights, and infant and young child mortality, (c) cause irreversible brain damage in fetus, infant and young children [12], and (d) effectuate short stature, delayed physical development, iodine-induced hyperthyroidism (IIH), and increased susceptibility to nuclear radiation [11]. Goiter increases risk of thyroid cancer, and a large goiter may obstruct trachea and esophagus [1]. All these disorders are known as iodine de ciency disorders (IDD). A total goiter rate (TGR) of ≥ 5% in school-age children of an area re ects the risk of developing IDD in the populations of that area [12]. Major strategies to control ID are implementing universal salt iodization (USI) and monitoring iodine intake periodically [11]. However, more than adequate and excess iodine intakes as evidenced by UI levels of 200-299 µg/L and ≥ 300 µg/L respectively may also cause hypothyroidism [13]. Excess iodine intake may also produce autoimmune thyroid diseases [11].
In the context of thyroid function, Tripura's tribal population has special relevance, because the State of Tripura is in the goiter endemic belt of India [14] and iodine de cient [15], and found goiter endemic [16][17][18][19]. Besides, effectiveness of prophylactic USI program introduced in the State prohibiting the sale of non-iodized edible salt on 31st August, 1988 [20] was not satisfactory [19]. Furthermore, Tripura's tribal population often consumes BS [21] rich in antithyroid constituents [22]. But information on the status of thyroid functions in the tribal population was found scanty. Accordingly, objectives of the study were to assess TV, thyroid pathophysiology, TH, TSH, Tg-Ab, TPO-Ab, UI, USCN, SI, WI, BS consumption pattern, and their inter-relationships in Tripura's tribal children.

Study Areas
Three hilly villages Deorachhara, Unakoti and Naithangchhara were randomly selected from Tripura Tribal Autonomous District Council. Deorachhara and Unakoti are in Unakoti district, and Naithangchhara is in Dhalai district. All villages are sparsely populated and under USI program, besides all are similar in climate, economy, culture, education, dietary habits and available health facilities.

Study Population
School-aged children are a useful target population for IDD's assessment and surveillance because of their ability to re ect IDD's current status [12]. Therefore, native-born children aged 6-12 years, who were free from diseases, were targeted from tribal populations who traditionally consume BS. In selected villages, 127 children (59 boys and 68 girls) were randomly selected from willing target children. The study was conducted during 2015-2018. Selected children's demographic characteristics have been shown in Table 1.

Thyroid Ultrasonography
Echogenicity of thyroid gland was studied for detecting nodules and measuring TV following WHO's ultrasonographic guidelines [11], using a portable compact Color Doppler Ultrasound Scanner System with a 7.5 MHz linear array transducer (GE LOGIQ E BT 12 Console with 12 L RS Linear Probe, Wipro GE Healthcare Pvt. Ltd) by a radiologist. Each thyroid lobe's volume (in milliliters) was calculated by the formula: 0.479 x (length in centimeters) x (width in centimeters) x (depth in centimeters). Thyroid volume was calculated by adding volumes of right and left lobes. Thyroid volume of each child was compared with 97th percentile reference TV of iodine-su cient children aged 6-12 years [11] to diagnose the child's thyroid gland as goitrous in case the child's TV exceeded the reference TV for the child's sex and age.

UI levels
Urine samples (each of about 50 ml) were collected from the selected children in wide-mouthed, screw capped, plastic bottles (adding a drop of toluene to each) and stored at 4 o C until analysis. In all, 127 urine samples were collected from three study villages ensuring at least 40 samples from each village, as urine samples collected at random from 40 subjects of an area represent the iodine nutrition status of that locality [23]. Iodine content of collected urine samples were assayed following the method of Karmarkar et al [24]. The procedure included: taking 0.1 ml of each urine sample, double distilled water (for a blank) and each working iodine standard in separate test tubes; adding 0.3 ml of 2.5 N potassium carbonate; drying overnight at 80 to 100°C; ashing in mu e furnace at 600 o C for 2 hours; adding 3 ml of 0.003 N sodium meta-arsenite on cooling; centrifuging at 2500 g for 7 minutes; incubating supernatant for 5 minutes at 56 o C; adding 3 ml of 0.005 N ceric ammonium sulphate at 30 second intervals; mixing; after 20 minutes recording transmittance at 420 nm; and determining urinary iodine by interpolation from standard curve. Estimation of iodine contents of urine samples was completed within 7 days of collection.

USCN levels
The dietary intake of thiocyanate in a population is determined from the daily urinary excretions and urinary concentrations of thiocyanate [25]. Thiocyanate contents of 127 collected urine samples (ensuring at least 40 samples from each village) were estimated following the method of Aldridge [26] as modi ed by Michajlovskij and Langer [27]. Analyses of thiocyanate contents of urine samples were done within 7 days of collection.

SI levels
Iodized salt samples (each of about 50 g) were collected from the families of the all selected children in the marked screw capped wide mouthed plastic bottles, ensuring at least 35 salt samples from each village, as 35 salt samples collected randomly from an area can re ect the status of USI in that area [28]. Collected salt samples were stored in cool and dark places until analysis. Iodine contents of iodized salt samples were estimated following iodometric titration method [11] within 7 days of collection.

WI levels
Samples (each of about 50 ml) of water used for drinking and house-hold works were collected in wide-mouthed, screw capped, plastic bottles from the families of all children and stored at 4 o C until analysis. Water samples were assayed following the method of Karmarkar et al [24]. The method involved: taking 7 ml of each water sample, double distilled water (for a blank) and each working iodine standard in separate test tubes; adding 1 ml of 20% sodium chloride, 0.5 ml of 60% sulphuric acid and 0.5 ml of 0.1 N arsenous acid; mixing; keeping in a water bath at 30 o C; adding 1 ml of ceric ammonium sulphate; exactly after 20 minutes adding 1 ml of ferrous ammonium sulphate (to stop the reaction) and 0.5 ml of 4% potassium thiocyanate; recording transmittance at 550 nm; and determining WI by interpolation from standard curve. Iodine contents of water samples were analyzed within 7 days of collection.

BS Consumption
To determine per capita daily BS consumption in selected villages, 61 randomly selected ST families consisting of 270 members were interviewed regarding their consumed BS's types and forms, duration of BS availability in a year, consumption frequency per week, consumption amount per serve, number of family members, and the information thus obtained was recorded.

Ethical Clearance
The study got approval from the Institutional Human Ethical Committee, Department of Physiology, University of Calcutta, Kolkata, India (IHEC/PHY/CU/H-P 34/13, dated 3rd October, 2013). Informed consents of the studied children were obtained in writing from their guardians.

Statistical Analysis
Mean ± SDs, medians and IQRs of age, TV, FT 4 , FT 3 , TSH, Tg-Ab, TPO-Ab, UI, WI, SI, USCN and BS consumption were calculated using Microsoft Excel 2007. Two tail t-test analysis of 'Two Samples assuming Unequal Variance tool' of Microsoft Excel Analysis ToolPak (MSEATP) was used to calculate mean differences' signi cance levels (p-value). To obtain correlation coe cients (r) as multiple R and their p-value, and to prepare regression lines of Fig. 1, MSEATP's 'Regression Analysis Tool' was used.

Results
Mean ± SDs, medians and IQRs of age, TV, FT 4 , FT 3 , TSH, Tg-Ab, TPO-Ab, UI, USCN, SI and WI are shown by villages and total in Table 1. All children had normal TV and 0.8% children had a thyroidal nodule. Table 1 Demographic characteristics, and levels of TV, FT 4 , FT 3 , TSH, Tg-Ab, TPO-Ab, UI, WI, SI, USCN and BS consumption by village and overall, and signi cance levels of differences between the means of the villages in tribal children of Tripura * Signi cance levels of differences between the means of the concerned villages for all variables at α = 0.05 of two-tail t-test.
There were no signi cant differences between means of boys and girls for the variables ( Table 2). Table 2 Age, and levels of TV, FT 4 , FT 3 , TSH, Tg-Ab, TPO-Ab, UI, WI, SI and USCN by sex and signi cance levels (p-value) of difference between the means of boys and girls in tribal children of Tripura * Signi cance level of difference between the means of boys and girls at α = 0.05 of two-tail t-test. Table 3 Distribution of levels of TSH, FT 4 , FT 3 , Tg-Ab, TPO-Ab, UI, WI and SI under different ranges in tribal children of Tripura Table 4 Correlation coe cients (r) with the levels of signi cance (p-value) for different variable pairs related to thyroid functions by sex and overall in tribal children of Tripura*

Discussion
The study reveals the status of thyroid functions in BS consuming Tripura's tribal children assessing their TV, thyroid pathophysiology, TH, TSH, Tg-Ab, TPO-Ab, UI, USCN, SI, WI and BS consumption.
WI and its relation with UI Bioavailability of iodine in an area is generally re ected in its WI. An area is considered as a severe, moderate or relative iodine de cient zone or iodine su cient zone depending on whether WI content is < 4, 4-10, > 10-20 or > 20 µg/L respectively [29]. As per the criteria, studied villages were in severe to moderate iodine de cient zones, in overall being severe iodine de cient (Table 1) and found consistent with our previous report [15]. However, there was a positive correlation between WI and UI (r = 0.2039, p = 0.021) in the studied children (Table 4) as observed also by Lv et al [30]. All these suggest that though the studied villages were iodine de cient, foodstuffs available in the villages were signi cant sources of the consumed iodine.

SI and its relation with UI
Earlier studies have reported both positive [19] and no [30] associations between SI and UI; however, the present observations are found consistent with the views of the latter. Besides, the ongoing USI program has yet to achieve the recommended adequacy level of ≥ 15 ppm iodine in ≥ 90% salt samples [11]. However, the USI program under present study was better in effectiveness than that of 1996-98 showing iodine adequacy level in about 63% salt samples [19] and, all studied villages had WHO/UNICEF/ICCIDD recommended median UI levels of 100-199 µg/L [11] indicating adequate iodine nutrition in the population of studied villages. In all, 54.3% of children studied had UI levels of ≥ 100 µg/L, and 45.7% had UI levels of < 100 µg/L (Table 3) which may be explained by the differences in hydration rather than by true ID [31]. All these indicate that the introduced prophylactic USI program was not effective enough in the villages and consequently was not a signi cant source of consumed iodine for studied children.

UI and its relations with TSH, FT 4 and FT 3
Previous studies on the association of iodine intake level with the levels of T 4 , T 3 and TSH con ict with each other and thereby suggest no clear relation of iodine intake level with the levels of T 4 , T 3 and TSH [13,[32][33][34][35].
In present study, UI was positively related with TSH (r = 0.2633, p = 0.003) which, in turn, remained positively associated with FT 4 (r = 0.2135, p = 0.016) and FT 3 (r = 0.1894, p = 0.033) ( Table 4) indicating TSH-dependent increase in TH by consumed iodine in studied children. A positive relation of UI with FT 4 (r = 0.2598, p = 0.032) in girls (Table 4) indicates that consumed iodine also increased FT 4 independent of TSH in girls. The nding as mentioned was found consistent with the views of an in vitro study that iodine increases TH, stimulating tyrosine iodination and coupling reactions, by binding to a limited number of high-a nity sites on TPO [36].

TSH and its relations with FT 4 and FT 3
Means of FT 4 and FT 3 as noted (Table 1) were higher and lower respectively than that of a study with excessive iodine intake [32], and higher in both cases than that of a study having adequate iodine intake [33]. Median TSH as observed (Table 1) was lower than that of the studies with excessive iodine [32], and adequate iodine [33]. Low FT 4 levels (hypothyroxinemia) and high FT 4 levels (hyperthyroxinemia) were observed in 2.4% and 4.7% children respectively (Table 3), and remained associated with normal TSH levels. Low FT 3 levels (hypotriiodothyroninemia) and high FT 3 levels (hypertriiodothyroninemia) were observed in 1.6% and 17.3% children respectively (Table 3), and remained associated with normal TSH levels. The possible cause of higher hypertriiodothyroninemia prevalence as observed may be for TSH's positive association with FT 3 . It has been also reported that high-carbohydrate diets remain positively associated with higher triiodothyronine levels, and higher triiodothyronine levels were related to increased iodine requirement. Increased iodine requirement exceeding iodine availability may cause IDD [37]. Low TSH levels (hypothyrotropinemia) and high TSH levels (hyperthyrotropinemia) were found in 4.7% and 1.6% children respectively (Table 3), and remained associated with normal FT 4 and FT 3 levels. Overall prevalence of hyperthyrotropinemia in present study ( were below their set points which led to compensatory increase in TSH that subsequently raised the levels of FT 4 and FT 3 in studied children. Further, positive association between FT 4 and FT 3 (r = 0.2006, p = 0.024) as observed ( Thyroid gland and its relation with age and FT 4 Thyroid gland is considered goitrous when its volume exceeds 97th percentile thyroid volume of iodine-replete population, and TGR of 0.0-4.9, 5.0-19.9, 20.0-29.9 and ≥ 30% indicate 'no', 'mild', 'moderate' and 'severe' IDD respectively [11]. Besides, prevalence of thyroid nodules in children may generally vary within 1.0-1.5% [40]. Present study showing all children with normal TV and 0.8% children with a thyroidal nodule suggests that IDD is not a major problem in studied villages. However, the present study's TV (Table 1) was lower than that of the excess iodine-exposed area where IDD based on TGR was a moderate public health problem [32]. A cohort study reported that TV was correlated positively with FT 4 in both sexes' euthyroid adults, and negatively with age in females [41]. However, positive associations of present study's TV with age (r = 0.5497, p = < 0.001) and FT 4 (r = 0.2466, p = 0.005) ( Table 4) suggest that TV increased with age, and increase in TV elevated FT 4 in studied children.
Thyroid autoantibodies and their relations with UI, USCN, TSH and FT 4 Literature showing both excessive UI and low UI as risk factors [33] and no risk factors [42] for thyroid autoimmunity suggests no clear association of excessive UI and low UI with thyroid autoimmunity. The prevalence of thyroid autoimmunity in children varies in between 0.5-3% in areas having no IDD [42]. However, more than adequate and excessive iodine intake may convert the state of higher Tg-Ab or TPO-Ab to overt hypothyroidism [13]. Median Tg-Ab as found (Table 1) was lower than that of adequate iodine nutrition zone [33], and the prevalence of positive Tg-Ab and positive TPO-Ab (Table 3) were lower than that of excess iodine area [32]. Negative associations of Tg-Ab as observed with TSH (r=-0.2024, p = 0.023) and FT 4 (r=-0.1869, p = 0.035) (Table 4) suggest that Tg-Ab increased the risk of central hypothyroidism while positive association of Tg-Ab with TPO-Ab (r = 0.3768, p = < 0.001) (Table) as found further indicates that Tg-Ab and TPO-Ab levels were related in such a way that they increased each other in studied children.
Besides, thiocyanate in excess may cause or aggravate endemic goiter in both iodine de cient [43] and iodine su cient regions [44]. It has been reported further that in ID, higher serum thiocyanate ( 1 mg/dl: at which thiocyanate inhibits iodine pump) associated with USCN of 1.9 mg/dl remain associated with lower T 4 and higher TSH, and thyroid hyperplasia [43]. However, the present study showing USCN's positive relation with FT 4 (r = 0.2477, p = 0.005) ( . Bamboo-shoots are found to increase TV and UI excretion, to reduce TPO activity, T 4 and T 3 levels in rats [49], and to decrease major enzyme activities of thyroid hormone synthesis in cultured thyrocytes [50]. Present study showing (i) children's maximal FT 4 in Deorachhara where consumption of BS was lowest and intake of iodine was adequate (Table 1), and (ii) children's maximal levels of TSH and FT 3 in Unakoti where BS consumption was highest and iodine intake was adequate (Table 1) -suggests that BS might have interfered with thyroid function in studied children. However, it has already been reported that the antithyroidal action of BS can be mitigated partially by iodine [49,51].
On the whole, it may be concluded that Tg-Ab tends to reduce the levels of both FT 4 and TSH in the children having adequate iodine nutrition, and in this condition (i) FT 4 and FT 3 remain up-regulated by TSH upsurge which is associated with rise in iodine intake, (ii) Tg-Ab is enhanced by TPO-Ab and may be increased by thiocyanate through TPO-Ab and (iii) consumed BS probably induces iodine-dependent elevation of TSH, and possibly contributes to the levels of FT 3 and thiocyanate in studied children.
Thus, it appears that studied children are at risk of developing hypothyroidism that may partially be reduced by decreasing BS consumption.