Iodine Status among Subclinical and Overt Hypothyroid Patients by Urinary Iodine Assay: A Case-Control Study.

OBJECTIVES: The objective of the study was to assess the differences of iodine status as measured by urinary iodine excretion (UIE) between cases of hypothyroidism and healthy controls.
MATERIALS AND METHODS: The study was conducted in cases with subclinical hypothyroidism (n = 58) and overt hypothyroidism (n = 41) and compared with age- and sex-matched healthy euthyroid controls (n = 52) attending Universal College of Medical Sciences Teaching Hospital, Bhairahawa, Nepal. Serum free triiodothyronine (fT3), free thyroxine (fT4), and thyroid-stimulating hormone (TSH) were estimated by competitive ELISA and sandwich ELISA, respectively (Diametra, Italy). The urinary iodine concentration (UIC) in urine samples was estimated by ammonium persulfate digestion method recommended by the WHO.
RESULTS: A significantly higher median UIC was observed among cases of subclinical hypothyroidism (224.90 μg/l) and overt hypothyroidism (281.0 μg/l) as compared to the controls (189.90 μg/l) (P = 0.0001, P = 0.001). Serum TSH in the cases of subclinical hypothyroid was higher, whereas fT3 was lower as compared to controls (P = 0.028, P = 0.0001), respectively. Similarly, serum TSH in the cases of overt hypothyroid was higher and fT3 and fT4 were lower as compared to controls (P = 0.0001, P = 0.0001, P = 0.015), respectively. There was positive correlation of UIC with TSH (r = 0.269, P = 0.0001), whereas negative correlation was seen with fT3 (r = -0.328, P = 0.0001) and fT4 (r = -0.145, P = 0.076). The test of multiple regression has shown that fT3 (β = -0.262, P = 0.012) as an independent predictor in association with UIE in cases.
CONCLUSION: Excessive iodine intake was found in hypothyroid patients as assessed by UIE concluding that it may trigger the thyroid hypofunction. Cohort studies to generate further evidence should be done to explore potential mechanism of hypothyroidism in excess iodine intake.

INTRODUCTION

Iodine is an essential trace element required for thyroid hormones synthesis.[12] Both iodine deficiency and excess intake may lead to thyroid dysfunction. Since 90% of ingested iodine is excreted in the urine, it is considered as a sensitive marker of current iodine intake.[3]

Excess iodine may decrease the release of thyroid hormone.[456] The use of USI in developing countries and the possible population health implications associated with excess iodine intake, there is an urgent need for better knowledge pertaining to this issue.[7] Hence, the present study can be the baseline data to show the impact of iodine status in hypothyroidism.

MATERIALS AND METHODS

Study design and sampling method

This study was carried from January 2016 to June 2016 in the Department of Biochemistry with collaboration of the Department of Internal Medicine at Universal College of Medical Sciences (UCMS), Bhairahawa, Nepal. Blood and urine samples were collected from all the participants of age group ≤50 years. Serum was separated and was stored at −20°C until evaluation. Free triiodothyronine (fT3), free thyroxine (fT4), and thyroid-stimulating hormone (TSH) were estimated by ELISA using Diametra kits, Italy. Ten milliliters spot urine samples in tightly capped plastic vials and samples were brought to the laboratory and kept at 4°C till analyzed. Urinary iodine concentration (UIC) in urine samples was estimated by the WHO recommended ammonium persulfate method. The consent was taken from each subject and the ethical approval for the study was provided by Institutional Review board of UCMS, Bhairahawa.

Urinary iodine analysis

Iodide is the catalyst in the reduction of ceric ammonium sulfate (yellow) to cerrous form (colorless) and is detected by the rate of color disappearance (Sandell-Kolthoff reaction).[8]

250 μl of each urine samples and series of standards were pipetted into the different test tubes followed by the addition of 1 ml of ammonium persulphate (1.0 mol/L) solution. All tubes were heated for 60 min at 100°C in heating block and then cooled to room temperature. After digestion, 2.5 ml arsenious acid solution (0.025 mol/) was added and mixed well. Then, it was kept for 15 min. 300 μl of ceric ammonium sulfate solution (0.038 mol/L) was added to each tube (quickly mixing) at 15–30 s intervals between successive tubes. It was allowed to sit at room temperature. Exactly 30 min after addition of ceric ammonium sulfate to the first tube, its absorbance was read at 420 nm. Successive tubes were read at the same interval as when adding the ceric ammonium sulfate.

Diagnostic criteria for hypothyroidism

As per the serum TSH profile, the thyroid disorders were classified as euthyroidism when the value of TSH was within the normal range (0.3–6.16 mIU/l), subclinical hypothyroidism if serum TSH was high (>6.16 mIU/l) but normal thyroid hormones, primary hypothyroidism, if serum TSH was >6.16 mIU/l and increased thyroid hormones.

Criteria for assessing iodine nutrition based on median urinary iodine (MUI) concentrations

The levels of iodine were defined according to the WHO/UNICEF/ICCIDD criteria[8] which are as follows:

Quality control

Quality control (QC) of procedures was done with appropriate standards along with precision and sensitivity of respective tests [Tables 1 and 2].

Table 1

Precision and sensitivity of hormonal assay

Table 2

Intra- and inter-assay coefficient of variation of urinary iodine assay

Data analysis

All the data were analyzed by Statistical Package for Social Service (SPSS) for Window version; SPSS 20, Inc., Chicago, IL, USA (IBM). The urinary iodine level was expressed as a median and compared by Mann–Whitney test. The other variables were expressed in terms of frequency (%), mean ± standard deviation, and compared by ANOVA or Student's t-test wherever applicable. Correlation between fT3, fT4, TSH, and UIC was analyzed with Pearson's correlation coefficient. P <0.05 was considered to be statistically significant.

RESULTS

In this case–control study, 58 Medicine Outpatients Department cases with primary hypothyroidism and 41 with overt hypothyroidism were compared with age- and sex-matched 52 euthyroid healthy controls.

The prevalence of excessive iodine intake in subclinical hypothyroidism was 17.3% and in overt hypothyroidism was 36.6%. In total population, 60.3% of patients with subclinical hypothyroidism, 46.3% of patient with overt hypothyroidism, and 36.5% of control exhibited higher than optimal UIC [Table 3].

Table 3

Iodine nutrition status in cases and controls based on urinary iodine excretion

Higher median urinary iodine (MUI) was noted in subclinical hypothyroid cases (224.9 μg/l) than controls (189.9 μg/l) that was statistically significant (P = 0.0001). Higher body mass index (BMI), serum TSH was observed and lower fT3 was observed in cases as compared to control (P = 0.03, P = 0.028, P = 0.0001), respectively. Systolic blood pressure (BP), diastolic BP, pulse, height, and fT4 level did not differ between cases and controls (P = 0.848, P = 0.992, P = 0.167, P = 0.984, P = 0.115). There was no significant difference in mean age (P = 0.721) [Table 4].

Table 4

Comparison of demographic and biochemical parameters in case (subclinical hypothyroidism) and control

Similarly, higher MUI level was noted in overt hypothyroid cases (281.0 μg/l) than controls (189.9 μg/l) that was statistically significant (P = 0.0001). Higher BMI and serum TSH were observed and lower fT3 and fT4 were observed in cases as compared to control (P = 0.024, P = 0.0001. P = 0.001, P = 0.015), respectively. Systolic BP, diastolic BP, pulse, and height did not differ between cases and controls (P = 0.719, P = 0.616, P = 0.366, P = 0.662). There was no significant difference in mean age (P = 1.000) [Table 5].

Table 5

Comparison of demographic and biochemical parameters in case (overt hypothyroidism) and control

There was positive correlation of urinary iodine excretion (UIE) with serum TSH level (r = 0.269, P = 0.0001), age (r = 0.018, P = 0.825), BMI (r = 0.009, P = 0.917), systolic BP (r = 0.009, P = 0.917). UIE was negatively correlated with fT3 (r = −0.328, P = 0.0001), fT4 (r = −0.145, P = 0.076), and diastolic BP (r = −0.084, P = 0.302) [Table 6].

Table 6

Pearson's correlation of urinary iodine level with different parameters

There were significant association of serum fT3, TSH levels, and BMI with various iodine nutrition status (P = 0.0001, P = 0.0001, P = 0.031) [Table 7].

Table 7

Association of various iodine nutrition status among variables (thyroid function test and body mass index)

We did not observe significant association of serum fT3, fT4, and TSH levels and BMI with cutoff UIE 100 μg/l (P = 0.881, P = 0.812, P = 0.983). However, we observed significant association of serum fT3, fT4, and TSH levels at cutoff UIE 300 μg/l (P = 0.0001, P = 0.024, P = 0.001) [Tables 8 and 9].

Table 8

Association of iodine status among variables (thyroid function test and body mass index) based on urinary iodine excretion at cutoff urinary iodine excretion 100 μg/L

Table 9

Association of iodine status among variables (thyroid function test and body mass index) based on urinary iodine excretion at cutoff urinary iodine excretion 300 μg/L

The test of independency has shown that fT3 (β = −0.262, P = 0.012) as an independent predictor in association with UIE in the hypothyroid case [Table 10].

Table 10

Urinary iodine excretion and its association as an independent predictor of hypothyroidism

DISCUSSION

In this study, MUI concentration was higher in the cases with subclinical hypothyroidism (224.9 μ/l) and overt hypothyroidism (281.0 μg/l) as compared to controls (189.9 μg/l) (P = 0.0001, P = 0.0001) which was in agreement with study done by Kotwal et al.[7] Another study conducted by Zhao et al.[9] reported that the MUI concentration was 184.5 μg/l and 169.6 μg/l for case group and control group, respectively (P = 0.003). Based on their report, high iodine intake was likely to lead to the occurrence of thyroid diseases, through a long-term mechanism and individual UIC detection was recommended for the disequilibrium of the iodine nutritional status among normal people.

In the current study, patients with hypothyroidism have a significant positive correlation between UIC and TSH levels. In agreement with our result, Konno et al.[10] reported that high UIC significantly correlated with subclinical hypothyroidism. They concluded that the prevalence of hypothyroidism in iodine sufficient areas may be associated with the amount of ingested iodine and the excessive iodine intake should be considered an etiology of subclinical hypothyroidism in addition to chronic thyroiditis.

Similar results were reported by Shan et al.[11] They demonstrated that elevating iodine intake reflected by excess UIC could increase the risk for the development of overt and subclinical hypothyroidism. Hall and Lazarus reported that iodine excess intake could increase the prevalence of autoimmune thyroid diseases.[12]

To our knowledge, this study was the first to show that excessive iodine was associated with hypothyroidism among adults of Southwestern Nepalese. In our study, we observed that there were significant association of serum fT3, TSH levels, and BMI with various iodine nutrition status (P = 0.0001, P = 0.0001, P = 0.031), respectively. Therefore, there was significant relationship among iodine nutrition, thyroid hormone profile, and dyslipidemia with hypothyroidism. We also observed significant association of serum fT3, fT4, and TSH levels at cutoff excess UIE with cutoff 300 μg/l (P = 0.0001, P = 0.024, P = 0.001), respectively, but we did not observe significant association with cutoff UIE 100 μg/l (P = 0.881, P = 0.812, P = 0.983), respectively [Tables 8 and 9]. Hence, our result suggests that excess iodine intake may play a role in the development of hypothyroidism.

Our study revealed that 60.3% of patients with subclinical hypothyroidism, 46.3% of patients with overt hypothyroidism, and 36.5% of control exhibited higher than optimal UIC. However, UIC was higher on patients than controls, the full cause of excessive iodine excretion remains unknown. This could be explained by difference in individual iodine intake. This pays our attention to excessive iodine exposure of patients and controls. The iodine concentrations in iodized salt may exceed the production level concentration of 20–40 ppm recommended by the WHO,[13] this excess exposure unmasks the thyroid dysfunction in susceptible individuals.

The mechanism behind the iodine-induced hypothyroidism is unknown, but several mechanisms could be involved. Iodine has been associated with thyroid autoimmunity.[14] As studied in vitro systems, excess iodine may lead to apoptosis of thyroid follicular cells and endothelial cells leading to thyroglobulin accumulation in connective tissue.[1516] Iodine supplementation also affects other aspects of thyroid health. In genetically susceptible mice, high iodine uptake has been reported to initiate and exacerbate infiltration of thyroid by lymphocytes[17] and may worsen the uptake defect of iodine thus decreasing the thyroid hormone synthesis.[18]

Hence, the comorbidity associated with hypothyroidism can be identified by evaluating UIC with certain other parameters. Moreover, there might be interplay between hypothyroidism and excess UIE in progression of severity and pathophysiological basis of thyroid dysfunction.

CONCLUSION

In our study, excessive iodine intake was found in hypothyroid patients. This may conclude that the high iodine intake may trigger the thyroid hypofunction. This study recommends the reappraisal of salt iodization campaign to limit the iodine excess and development of hypothyroidism. However, further studies are warranted to confirm our results and to generate further evidence that would explain the potential mechanism of hypothyroidism in excess iodine intake.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.