Predictors of Change in Serum TSH after Iodine Fortification: An 11-Year Follow-Up to the DanThyr Study

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1 ORIGINAL Endocrine ARTICLE Care Predictors of Change in Serum TSH after Iodine Fortification: An 11-Year Follow-Up to the DanThyr Study Lena Bjergved, Torben Jørgensen, Hans Perrild, Allan Carlé, Charlotte Cerqueira, Anne Krejbjerg, Peter Laurberg, Lars Ovesen, Inge Bülow Pedersen, Lone Banke Rasmussen, and Nils Knudsen Research Centre for Prevention and Health (L.B., T.J., C.C.), The Capital Region of Denmark, DK-2600 Glostrup, Denmark; Department of Endocrinology and Gastroenterology (L.B., H.P., N.K.), Bispebjerg University Hospital, DK-2400 Copenhagen, Denmark; Department of Endocrinology and Medicine (A.C., A.K., P.L., I.B.P.), Aalborg Hospital, Aarhus University Hospital, and Faculty of Medicine (T.J., P.L.), Aalborg University, DK-9000 Aalborg, Denmark; Department of Gastroenterology (L.O.), Slagelse Hospital, DK-4200 Slagelse, Denmark; Division of Nutrition (L.B.R.), National Food Institute, Technical University of Denmark, DK-2860 Søborg, Denmark; and Faculty of Health Sciences (T.J.), University of Copenhagen, DK-2200 Copenhagen, Denmark Context: Few data are available on the effect of iodine fortification on thyroid function development in a population. Objective: Our objective was to evaluate changes in thyroid function after iodine fortification in a population and to identify predictors for changes in serum TSH. Design and Setting: A longitudinal population-based study of the DanThyr C1 cohort examined at baseline ( ) and reexamined 11 yr later ( ). The mandatory program for iodization of salt was initiated in Participants: A total of 2203 individuals, with no previous thyroid disease, living in two areas with different levels of iodine intake, with measurement of TSH and participation in follow-up examination were included in the analysis. Main Outcome Measure: Change in serum TSH was evaluated. Results: During the 11-yr follow-up, mean TSH increased significantly from 1.27 mu/liter [95% confidence interval (CI) ] to 1.38 mu/liter (CI ) (P 0.001). The most pronounced increase was observed in the area with the highest iodine intake [1.30 mu/liter (CI ) to 1.49 mu/liter (CI ), P 0.001], whereas the increase was not significant in the low-iodine-intake area [1.24 (CI ) to 1.28 (CI ), P 0.06)]. Change in TSH was positively associated with the presence of thyroid peroxidase antibody at baseline (P 0.001) and negatively associated with baseline thyroid enlargement (P 0.001) and multiple nodules (P 0.001). Conclusions: Even small differences in the level of iodine intake between otherwise comparable populations are associated with considerable differences in TSH change at the 11-yr follow-up. Multinodular goiter predicted a less pronounced TSH increase during follow-up, which may be explained by iodine-dependent activity of autonomous nodules. (J Clin Endocrinol Metab 97: , 2012) ISSN Print X ISSN Online Printed in U.S.A. Copyright 2012 by The Endocrine Society doi: /jc Received June 15, Accepted August 15, First Published Online September 7, 2012 Abbreviations: CI, Confidence interval; GEE, generalized estimating equations; TPO-Ab, thyroid peroxidase antibody jcem.endojournals.org J Clin Endocrinol Metab, November 2012, 97(11):

2 J Clin Endocrinol Metab, November 2012, 97(11): jcem.endojournals.org 4023 The level of iodine intake influences the occurrence and the pattern of thyroid diseases (1 6). Severe iodine deficiency leads to hypothyroidism associated with increased risk of pregnancy complications and impaired mental development in children, goiter, and increased occurrence of thyroid function abnormalities (7). Mild to moderate iodine deficiency may cause thyroid enlargement, goiter (especially in older age groups) (8), and hyperthyroidism (9, 10). Finally, iodine excess may lead to increased risk of hypothyroidism, thyroid autoimmunity, and a shift in types of thyroid cancer (11). It has been estimated that in 2007, 70% of the households throughout the world used iodized salt (12). Monitoring of fortification programs is essential to avoid both iodine excess as well as to correct its deficiency (13). The final objective of fortification programs is not only to normalize urinary iodine but to normalize thyroid function (14). However, few data are available on the effect of iodine fortification on thyroid function. The main goal of this study was to assess the impact of a nationwide iodine fortification program on the development of thyroid function in individuals with special emphasis on identifying determinants for a change in serum TSH. Subjects and Methods Design and population The Danish investigation of iodine intake and thyroid disease (DanThyr) is a comprehensive program monitoring the nationwide iodine fortification of household salt and salt used in the commercial production of bread in Denmark, described earlier in detail(15). The present study includes data from a cross-sectional populationbased study, conducted before iodization, and a follow-up study of participants. Detailed descriptions of the baseline study have been published previously(8). In brief, 9274 people of Danish nationality were invited to participate; they were drawn randomly from the two regions of Denmark, representing areas with mild (Copenhagen) and moderate (Aalborg) iodine deficiency, using the national Civil Registration system. In this system, all inhabitants of Denmark can be identified by a unique 10-digit number. The study population comprised women of four age groups from yr and men of one age group (60 65 yr). Of those invited to participate during , 4649 (50.1%) attended (9). Participants were invited for follow-up between February 22, 2008, and February 11, Seventy-two of the 4649 baseline participants had emigrated and 403 had died, leaving 4174 to be invited of whom 2465 participated (59.1%) (Fig. 1). Mean follow-up time was 11.2 yr (range, yr), and mean exposure time to mandatory iodization (implemented July 1, 2000) was 8.6 yr (range, yr). All procedures (e.g. invitation, questionnaires, and examination) were similar at baseline and follow-up examination. Participants underwent physical examination and gave a nonfasting venous blood sample and a spot urine sample, and a thyroid ultrasonography was performed (Siemens Sonoline Versa Pro; Siemens, Erlangen, Germany). The same two sonographers, each in Aalborg and Copenhagen, performed all the ultrasonographies at baseline and follow-up examinations using the same equipment. Thyroid volume, the number of distinct nodules, and the size of the nodules showed to be valid for comparisons between the two observers with minimal inter-observer variation, described earlier in details (16). Blood and urine samples were collected between 0800 and 1730 h, frozen ( 20 C), and subsequently analyzed in random order with respect to region, sex, age, and season of the year at examination, at baseline, and at follow-up. Reference ranges derived from the baseline cohort were defined as the following: TSH, mu/liter; free T 4, pmol/liter; and free T 3, pmol/liter (9). Before fortification, the median iodine excretion in spot urine samples was 68 g/liter in Copenhagen and 53 g/liter in Aalborg, increasing to 84 and 83 g/liter, respectively, at followup. Excluding individuals taking iodine supplements, the baseline median iodine excretion was 61 g/liter in Copenhagen (mild iodine deficiency according to the World Health Organization criteria) (12) and 45 g/liter in Aalborg (moderate iodine deficiency) and had increased to 76 and 74 g/liter, respectively, at follow-up (mild iodine deficiency in both regions). Of the investigated individuals, 34% took iodine-containing supplements at baseline, similar to the 36% taking them at follow-up. Laboratory methods TSH, free T 4, and free T 3 were analyzed at baseline with LUMItest assays (BRAHMS, Berlin, Germany) and with the Roche Modular E system by electrochemical luminescence using ELECSYSatfollow-up. Asubsampleof201bloodsamplesfromthe baseline examination was reanalyzed with the assay used at followup. A Bland-Altman plot showed very low interassay variation for TSH without marked irregularities (data not shown). The bias for TSH was modest, with a mean difference of mu/liter [95% confidence interval (CI) mu/liter]. Reanalysis of serum free T 4 showed higher values when analyzed with the follow-up assay [1.65 pmol/liter ( pmol/liter)], and a higher free T 3 [0.11 pmol/liter ( pmol/liter)], although less pronounced, was also found. RIA for measurement of thyroid peroxidase antibody (TPO-Ab) (DYNOtest anti-tpo; BRAHMS Diagnostica, Berlin, Germany) has been described in detail previously (17). At follow-up, automated antibody assays (KRYPTOR anti- TPO n ) were used, as described in detail previously (18). Spot urine iodine concentrations were determined at both examinations using the Cerium/Arsenite method after digestion by alkaline ashing, as previously described (19, 20). The analytical sensitivity of the assay was 2 g/liter. The iodine laboratory was certified by the U.S. Centers for Disease Control and Prevention EQUIP program. Definition of variables Smoking status at baseline and follow-up was categorized into four groups: 1) current smokers, 2) former smokers, 3) never smokers, and 4) occasional smokers. Change in smoking status was analyzed in four categories in the prediction model: 1) current smokers at baseline and follow-up (n 379), 2) individuals who had never smoked (n 844), 3) former smokers at baseline and follow-up (n 359), and 4) those who stopped smoking during follow-up (n 277). Few people started smoking during follow-up (n 26), and occasional smokers are known to be a very heterogeneous group (n 266); both groups were excluded in the analysis. TPO-Ab of 30 ku/liter or higher was considered antibody positive, according to the detection limits defined previously (17). Thyroid volume was calculated as maximal length width depth /6

3 4024 Bjergved et al. Change in Serum TSH after Iodine Fortification J Clin Endocrinol Metab, November 2012, 97(11): FIG. 1. DanThyr C1 flow diagram. of each lobe, and thyroid enlargement was defined as a thyroid volume over 18 ml for women and over 25 ml for men. Echogenicity was categorized into five groups: 1) markedly decreased, 2) slightly decreased, 3) normal, 4) slightly increased, or 5) markedly increased (compared with adjacent muscles); groups 1 and 2 were included in the definition of hypoechogenicity. Solitary nodules were not included in the definition of multiple nodules. The following categories were used in the questionnaire related to self-rated health ( How would you rate your health in general? ): very good, good, fair, bad, and very bad. Statistical analysis Data were analyzed using SAS version 9.2 (SAS Institute Inc., Cary, NC). Two-sided P 0.05 was considered significant. Nonnormally distributed variables were log transformed to approach normal distribution. The primary outcome measure was change in serum TSH from baseline to follow-up using TSH as a continuous variable. To estimate the adjusted change in TSH in correlated data, we used a multiple linear regression model with generalized estimating equations (GEE) and exchangeable correlation matrix. Possible confounding by age group, sex, and change in smoking status was considered. The age effect on change in TSH was analyzed in a linear regression model with GEE, adjusting for region (a proxy for iodine intakes), change in smoking status, and change in thyroid volume (continuous variable) during follow-up. Only women were included in this model. The assumption of linearity of age was tested by including age 2 in the model, which was not significant (P 0.21). Sex-related differences in TSH change were estimated by an interaction term between sex and period (time of measurement), in a multiple linear regression model with GEE, with TSH as outcome and adjusting for region, age, and change in smoking status. This analysis was limited to persons from yr of age. Another multiple linear regression model was used in analysis of the determinants of 11-yr TSH change. In this model, serum TSH at follow-up was used as outcome variable and TSH concentration at baseline as a predictor variable. Potential selection bias in follow-up was evaluated using information at baseline (Table 1). Comparisons of the baseline characteristics of participants at the follow-up examination with nonparticipants were done by 2 for categorical variables, t test for means, and Wilcoxon rank test for medians. Participants who reported current or previous treatment with medicine, surgery, or radioactive iodine therapy for thyroid disease at baseline or follow-up (n 228) and those missing at least one value of TSH (n 36, including two persons who received medical treatment) were excluded, leaving 2203 individuals for analysis. The study protocol was approved by the Danish Ethics Committee ( and VN 96/208mch and N-VN MCH, the Northern Danish Region committee), and the study was conducted in accordance with the Declaration of Helsinki. All participants gave written informed consent. Results TSH measures and predictors of TSH change During the 11 yr of follow-up, mean TSH increased significantly [from 1.27 mu/liter (95% CI ) to 1.38 mu/liter (CI ), P 0.01]. The most

4 J Clin Endocrinol Metab, November 2012, 97(11): jcem.endojournals.org 4025 TABLE 1. Baseline characteristics of members of the DanThyr C1 cohort, categorized by participation or nonparticipation at follow-up pronounced increase was observed for people living in Copenhagen [from 1.30 mu/liter (CI ) to 1.49 mu/liter (CI ), P 0.01], whereas for those living in Aalborg, the increase was not statistically significant [from 1.24 mu/liter (CI ) to 1.28 mu/ liter (CI ), P 0.06]. Overall, TSH increased in all age groups, although not significantly in women in the 18- to 22- and 60- to 65-yr-old ranges. Adjusting for age, sex, and change in smoking status in a multiple linear regression model taking correlation of data into account did not change the conclusions (Fig. 2). Analysis of determinants of TSH change showed that the presence of TPO-Ab at baseline was associated with a significantly steeper increase in serum TSH compared with their absence (Table 2). Individuals with a thyroid ultrasonography showing thyroid enlargement or multiple nodules at baseline had a significantly lower increase in serum TSH compared with those without. In individuals living in the region with the lowest iodine intake at baseline (Aalborg), a significantly lower TSH increase was shown compared with the region with the higher iodine intake (Copenhagen). No significant association was found between change in TSH and familial disposition, hypoechogenicity at ultrasonography, or change in smoking habits. Participants at follow-up examination (n 2465) Nonparticipants at follow-up examination (n 1709) P value a Age groups Women, yr 489 (19.8%) 434 (25.4%) 0.01 Women, yr 514 (20.9%) 391 (22.9%) Women, yr 657 (26.7%) 237 (13.9%) Women, yr 381 (15.4%) 366 (21.4%) Men, yr 424 (17.2%) 281 (16.4%) Body mass index (kg/m 2 ) 24.2 ( ) 24.3 ( ) 0.08 Hypertension (SBP 140 or DBP 90 mm Hg) 1,015 (41.3%) 730 (42.8%) 0.33 Daily smokers 793 (32.1%) 668 (39.1%) 0.01 High risk alcohol consumption ( 14/21 drinks/wk) 180 (7.3%) 141 (8.3%) 0.26 History of thyroid disease (treated subjects) 104 (4.2%) 84 (4.9%) 0.29 Family history of thyroid disease 507 (20.6%) 309 (18.1%) 0.12 Self-rated health bad or very bad 81 (3.3%) 93 (5.5%) 0.01 TPO-Ab 30 ku/liter 391 (16.1%) 224 (13.4%) 0.02 Tg-Ab 20 ku/liter 293 (12.1%) 157 (9.4%) 0.01 Urinary iodine excretion ( g/liter) Including persons taking iodine-containing supplements 61 (34 101) 63 (35 104) 0.26 Excluding persons taking iodine-containing supplements 52 (29 80) 55 (30 86) 0.02 Thyroid volume (ml by ultrasonography) 12.6 ( ) 12.2 ( ) 0.01 TSH (mu/liter) 1.3 ( ) 1.3 ( ) 0.40 Free T 4 (pmol/liter) Free T 3 (pmol/liter) Serum Tg ( g/liter) 12.2 ( ) 12.4 ( ) 0.14 Data are expressed as mean SD, median (interquartile range), or n (percent). DBP, Diastolic blood pressure; SBP, systolic blood pressure; Tg, thyroglobulin. a P values are for comparison between participants and nonparticipants. The 2 test was used for categorical variables, t test for means, and Wilcoxon rank test for medians of continuous variables. Individual changes in thyroid function The follow-up TSH levels for persons with serum TSH within the normal reference range at baseline are shown in Table 3. Individuals with a baseline TSH in the lower FIG. 2. Serum TSH change by region, adjusted for sex, age, and change in smoking. Values are 1.28 mu/liter (95% CI ) at baseline and 1.45 mu/liter (95% CI ) at follow-up for Copenhagen (P 0.01) and 1.21 mu/liter (95% CI ) at baseline and 1.25 ( ) at follow-up for Aalborg (P 0.14); n 2203, excluding participants who reported current or previous treatment with medicine, surgery, or radioactive iodine therapy for thyroid disease at baseline or follow-up (n 228) and those missing at least one value of serum TSH (n 36, including two persons who received medical treatment).

5 4026 Bjergved et al. Change in Serum TSH after Iodine Fortification J Clin Endocrinol Metab, November 2012, 97(11): TABLE 2. Predictors of TSH change at 11-yr follow-up (n 1458) Estimate P Age 0.03 Women, yr Women, yr Women, yr Women, yr (reference) 0 Region (proxy for iodine intake) 0.01 Aalborg Copenhagen (reference) 0 Change in smoking during follow-up a 0.18 Current smoker (baseline and follow-up) Former smoker (baseline and follow-up) Stopped smoking during follow-up Never smoker (reference) 0 TPO-Ab 30 ku/liter at baseline 0.01 Yes No (reference) 0 Hypoechogenicity by ultrasonography 0.83 at baseline Yes No (reference) 0 Thyroid enlargement (volume /25 ml by ultrasonography) Yes No (reference) 0 Multiple nodules at baseline 0.01 Yes No (including solitary nodules) 0 (reference) Familial disposition of thyroid disease 0.93 Yes No (reference) 0 The DanThyr C1 cohort (Denmark) was examined at baseline ( ) and reexamined 11 yr later ( ). Subjects treated for thyroid disease (n 228), subjects with no TSH measurements (n 36), and men (n 403) were excluded. Multiple linear regression model included serum TSH at follow-up as outcome variable and TSH at baseline, age, region, change in smoking status, TPO-Ab status, ultrasound hypoechogenicity, thyroid enlargement, multiple nodules, and familial disposition as predictor variables. The estimate defines the number of units of change in log TSH (y) in the specific class of the predictor (x) compared with the reference group. The reference groups are indicated. a Person who started smoking during follow-up (n 26) and occasional smokers (n 266) were excluded from analysis. tertile of the normal TSH reference range were more likely to have a below-normal TSH at follow-up (4.8%) than those with a TSH in the upper tertile (0.2%). Similarly, 10.5% of the persons with a baseline TSH in the upper tertile of the normal reference range developed a high TSH during follow-up, whereas it happened for only 0.5% of the persons with a baseline TSH in the lower tertile. Effect of age and gender on TSH change Age (continuous) was not associated with a significant TSH change after adjusting for region, change in smoking status, and change in thyroid volume (P 0.93). No association between sex and TSH change was observed even after adjusting for region, age, and change in smoking status (P 0.88). Sex did not interact with period in the linear regression model taking correlation of data into account and adjusting for smoking status and region (P 0.14). Nonresponse analysis Characteristics of the entire cohort followed from baseline to follow-up are shown in Table 4 and characteristics according to attendance in Table 1. Nonparticipating individuals who were alive during the reexamination differed significantly from participants. Overall, they had lower baseline thyroid volume, were more likely to be regular smokers, and had a higher frequency of bad or very bad self-rated health at baseline. There were no differences in levels of serum TSH, free T 4, free T 3, and treatment for thyroid disease at baseline (Table 1). Discussion In this longitudinal population-based study, we measured TSH in 2429 individuals before and after 8.6 yr of exposure to mandatory iodine in household salt and salt used in the commercial production of bread. A significant ageadjusted increase in mean serum TSH was found, and the most pronounced increase was seen in the region with the highest iodine intake at baseline. Increase in TSH was more pronounced among participants with TPO-Ab at baseline and participants without goiter and multiple nodules. Urine iodine excretion is the best indicator of iodine intake in a population (12). We found an increase in iodine intake from mild and moderate iodine deficiency to mild deficiency in both regions. The values were significantly lower than those observed in a Danish cohort studied in (21), suggesting that the iodine intake of the Danish population has decreased in the recent years. Additional studies are necessary to evaluate this in detail. To measure exactly how much of the observed TSH increase is explained by the increased iodine intake after fortification of salt, a control group without intake of fortified salt would be necessary. This was not possible in our setting. Our study design, however, made it possible to compare two regions of Denmark with similar populations but with different levels of iodine intake. We demonstrated a significant increase in serum TSH after iodine fortification, and the increase remained highly significant after adjusting for age, sex, and change in smoking habits.

6 J Clin Endocrinol Metab, November 2012, 97(11): jcem.endojournals.org 4027 TABLE 3. Development in TSH level in euthyroid, untreated persons from baseline to follow-up (n 2100) Baseline TSH level 0.4 TSH 1.0 mu/liter (n 600) 1.0 TSH 2.0 mu/liter (n 1090) 2.0 TSH 3.6 mu/liter (n 411) TSH < 0.4 mu/liter 0.4 < TSH < 1.0 mu/liter The difference between regions, with a more pronounced TSH increase in the region with the highest iodine intake at baseline, strongly indicates that changes in iodine intakes explain at least partly the TSH increase. The thyroid gland is able to compensate by enhancing or limiting the use of available iodine by many thyroidal processes (22, 23). This is important in understanding how iodine affects thyroid function. In moderate or mild iodine deficiency, the thyroid gland adapts by inducing hyperactivity, thereby keeping thyroid hormone levels within a normal range. This process may lead to follicular 1.0 < TSH < 2.0 mu/liter TSH level at follow-up 2.0 < TSH < 3.6 mu/liter 3.6 mu/ liter < TSH Treated subjects (current or previous) 29 (4.8%) 261 (43.5%) 261 (43.5%) 28 (4.7%) 3 (0.5%) 18 (3.0%) 11 (1.0%) 158 (14.5%) 626 (57.4%) 232 (21.3%) 36 (3.3%) 27 (2.5%) 1 (0.2%) 16 (3.9%) 161 (39.2%) 170 (41.4%) 43 (10.5%) 20 (4.9%) The DanThyr C1 cohort (Denmark) was examined at baseline ( ) and reexamined 11 yr later ( ). Subjects treated for thyroid disease at baseline (n 104), no measurements of TSH (n 36), and baseline TSH outside the normal reference range (n 282, including 57 persons without measurements of TSH or treatment at baseline) were excluded from the analysis. TABLE 4. proliferation and mutations causing multifocal autonomous growth and function (11). It is well documented that an increased iodine intake may lead to a transitory increased incidence of hyperthyroidism in people with autonomous nodules (24, 25), presumably caused by an increase in substrate for thyroid hormone production (25). This study showed that multinodular goiter predicted a less pronounced TSH increase in the follow-up period, which could be explained by an increase in thyroid hormone production in autonomous thyroid nodules. The incidence of iodine-induced hyperthyroidism is expected Clinical and biochemical characteristics of the reexamined population at baseline and follow-up Baseline ( ) (n 2465) Follow-up ( ) (n 2465) P value a Age groups Women, yr 489 (19.8%) 489 (19.8%) Women, yr 514 (20.9%) 514 (20.9%) Women, yr 657 (26.7%) 657 (26.7%) Women, yr 381 (15.5%) 381 (15.5%) Men, yr 424 (17.2%) 424 (17.2%) Body mass index (kg/m 2 ) 24.2 ( ) 25.4 ( ) 0.01 Daily smokers 793 (32.2%) 470 (19.6%) 0.01 History of thyroid disease (treated subjects) 104 (4.2%) 213 (8.7%) 0.01 Family history of thyroid disease 507 (20.6%) 572 (23.3%) 0.01 Thyroid enlargement ( 18/25 ml by ultrasonography) 468 (19.0%) 433 (17.6%) 0.01 Thyroid volume (ml by ultrasonography) 12.6 ( ) 12.5 ( ) 0.01 TSH (mu/liter) 1.3 ( ) 1.5 ( ) 0.01 Free T 4 (pmol/liter) 14.6 ( ) 15.8 ( ) 0.01 Free T 3 (pmol/liter) 5.2 ( ) 4.6 ( ) 0.01 Serum Tg ( g/liter) 12.2 ( ) 8.4 ( ) 0.01 TPO-Ab 30 ku/liter 391 (16.1%) 585 (23.9%) 0.01 Tg-Ab 20 ku/liter 293 (12.1%) 526 (21.5%) 0.01 Urinary iodine excretion ( g/liter) Including individuals taking iodine supplements 61 (34 101) 84 (47 133) 0.01 Excluding individuals taking iodine supplements 52 (29 80) 75 (42 113) 0.01 Data are expressed as median (interquartile range), or n (percent). Information about smoking status (affirmative answer to daily smoking at the examination time), thyroid disease (current or previous treatment with medicine, surgery, or radioactive iodine therapy for thyroid disease at baseline or follow-up), and familial disposition of thyroid disease (at least one first-degree relative with thyroid disease) was obtained by questionnaires. Tg, Thyroglobulin. a P values show differences between time points. Comparisons were done by McNemar s test for categorical variables and Wilcoxon signed rank test for continuous variables as observations were dependent.

7 4028 Bjergved et al. Change in Serum TSH after Iodine Fortification J Clin Endocrinol Metab, November 2012, 97(11): to resolve spontaneously within years (5) but could still be part of the explanation of the change. Other possible effects of increasing iodine intake are abnormal inhibition of the thyroid function in individuals with thyroid autoimmunity (3, 23, 26), apoptosis in follicular cells (27), and generally down-regulation of a number of thyroidal processes; however, these mechanisms are not fully understood in humans. Considering the frequent occurrence of thyroid autoimmunity in the Danish population (17), it seems conceivable that the increased iodine intake in both regions resulted in a more pronounced inhibition of the thyroid in people with thyroid autoimmunity resulting in serum TSH increase. This is supported by the strong association found in the present study between the presence of TPO-Ab at baseline and subsequent TSH increase. Individuals from the Copenhagen area had a higher iodine intake during the follow-up period (21). Therefore, a more pronounced iodine inhibition of the gland is expected, and the more pronounced increase in TSH might be a logical consequence. Moreover, a nationwide study of incident use of thyroid hormone replacement therapy in relation to the implementation of iodine fortification in Denmark demonstrated a higher increase in incidence rate in the region with the higher baseline iodine intake, suggesting a higher incidence of hypothyroidism in this area (6). Low-normal and high-normal serum TSH level at baseline seems to be predictive for development of follow-up TSH values lower than and higher than the normal reference range, respectively. Hence, the baseline TSH level matters, even when TSH values are within the normal reference range. The normal reference range is population specific, but the individual reference range is probably narrower (28). We found no significant age effect on TSH change. Knudsen et al. (29) found, in an analysis of baseline data of this study, a negative association between TSH and age, and this association remained true 7 yr later in a Danish cohort study of a comparable population (30). Decreasing TSH with age in an iodine-deficient area, however, could possibly be explained by increasing frequency of multinodular goiter with age and hence increased prevalence of hyperthyroidism and decreasing TSH. Based on our analysis of the age effect on TSH change, adjusted for thyroid volume (a proxy for multinodular goiter), it seems unlikely that the observed increase in TSH is a result of the 11-yr age increase in the cohort. We are aware of no other longitudinal studies of an adult general population introduced to iodized salt. Other longitudinal epidemiological studies of development in thyroid function, with stable iodine intakes in iodinesufficient populations (31 33), have been focusing on subclinical and/or overt hypothyroidism as outcome measures. One longitudinal study initiated 3 yr after introduction of iodized salt in China with a 5-yr follow-up has been published (26). The study compared incidences of hypothyroidism in three regions with different iodine intake and showed no difference in cumulative incidence of overt hypothyroidism, which might be explained by the short follow-up time, the long latency of hypothyroidism, and relatively few cases. Our study is observational and not a randomized trial, and hence, possible causal relationships should be interpreted with care. The relatively low participation rate, 59.1% of those invited, might have introduced selection bias. On the other hand, information about level of TSH, free T 4, free T 3, frequency of treatment for thyroid disease, and family history of thyroid disease was obtained at baseline and did not differ significantly between participants and nonparticipants. Conclusions After 8.6 yr of exposure to mandatory iodization of salt in a population, thyroid function seems to be affected. Comparable populations show a considerable difference in TSH change although the difference between the levels of iodine intake was small. Multinodular goiter predicted a less pronounced TSH increase during follow-up, which may be explained by iodine-induced hyperthyroidism. The importance of future monitoring of iodization programs worldwide is exposed in this study where the iodine intake seems to have decreased in recent years, despite an unchanged iodization fortification level. Acknowledgments Special thanks are due to Ingelise Leegard and René Fiege for carefully performing the ultrasonographies and the laboratory work, to Mathilde Svendstrup for help with data collection, and to all the participants of the study for their important contributions. Address all correspondence and requests for reprints to: Lena Bjergved, M.D., Department of Endocrinology and Gastroenterology, Bispebjerg University Hospital, Bispebjerg Bakke 23, 2400 Copenhagen NV, Denmark. lena.baudler. bjergved@regionh.dk. This work was supported by the Danish Medical Foundation; the 1991 Pharmacy Foundation; the North Jutland County Research Foundation; the Tømmerhandler Wilhelm Bangs Foundation; the Copenhagen Hospital Corp. Research Foundation; the Ministry of Food, Agriculture, and Fisheries; the Danish Agency for Science, Technology, and Innovation; and the King Christian and Queen Louise Jubilee Scholarship. Disclosure Summary: The authors have nothing to disclose.

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