SERUM Tg is an important marker of cancer recurrence

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1 /01/$03.00/0 The Journal of Clinical Endocrinology & Metabolism 86(8): Printed in U.S.A. Copyright 2001 by The Endocrine Society Serum Tg A Sensitive Marker of Thyroid Abnormalities and Iodine Deficiency in Epidemiological Studies NILS KNUDSEN, INGE BÜLOW, TORBEN JØRGENSEN, HANS PERRILD, LARS OVESEN, AND PETER LAURBERG Department of Internal Medicine I (N.K., H.P.), Bispebjerg University Hospital, 2400 Copenhagen; Centre for Preventive Medicine (N.K., T.J.), Glostrup University Hospital, 2600 Copenhagen; Department of Endocrinology (I.B., P.L.), Aalborg Hospital, 9100 Aalborg; Institute of Food Research and Nutrition (L.O.), The Danish Veterinary and Food Administration; and The Danish Centre for Prevention of Thyroid Disease (Danthyr) 2860 Soøberg, Denmark Serum Tg is widely used in the control of thyroid cancer but also in the diagnosis of certain other thyroid diseases. Serum Tg may be useful in the characterization of the iodine status of a population, but little is known about determinants of serum Tg levels. We examined a random selection of 4,649 subjects from 2 regions in Denmark with different iodine status. Thyroid volume and structure were determined with ultrasonography, and thyroid function tests and Tg analysis were performed. The factor with the closest association with serum Tg levels was thyroid volume at ultrasonography (P < 0.001). Also thyroid nodularity (P < 0.001) and iodine excretion (P < 0.001) had close associations to serum Tg, even after adjusting for Abbreviations: Ab, antibodies; ID, iodine deficiency. the influence of the other parameters. Thyroid dysfunction had a less pronounced but still highly significant association with serum Tg (P < 0.001), but no relation was found to serum TSH in general. The association with age seemed to rely on differences in the prevalence of thyroid abnormalities, and men had lower Tg levels than women of the same age. There was a marked difference in serum Tg between the two regions with slightly different iodine excretion also after adjusting for the other factors. In conclusion, serum Tg reflects thyroid abnormalities and thyroid function and is a sensitive marker of iodine deficiency in a population. (J Clin Endocrinol Metab 86: , 2001) SERUM Tg is an important marker of cancer recurrence among patients who have received ablative therapy for thyroid cancer (1, 2). The value in the diagnosis of other thyroid diseases is limited, because serum Tg is increased in almost all kinds of thyroid disease with an overlap to healthy individuals. Still, some clinical applications of serum Tg measurements have been suggested, as for instance in the diagnosis of subacute thyroiditis (3), thyrotoxicosis factitia (4, 5), or as a marker of postoperative recurrence of nontoxic goitre (6). Serum Tg levels may predict the outcome of antithyroid drug therapy in Graves disease (7 9), whereas the ability of serum Tg measurements to predict the outcome of thyroid hormone treatment of nontoxic goitre has not been convincing (10, 11). Few studies have elucidated the epidemiology of serum Tg, and comparison of results from different studies is hampered by differences in the performance of the assays, interference with endogenous Tg antibodies (Tg Ab), and a lack of standardization, as reviewed by Spencer et al. (2). Consequently, generally accepted normal values of serum Tg have not been published, and comparisons between studies have been difficult. In comparative studies from areas with different iodine status, lower serum Tg values were reported in iodine-sufficient than in iodine-deficient areas (12 14), and serum Tg has been suggested as a marker of iodine deficiency disorders in a population (15 18). In the present study, we characterized serum Tg in a population and investigated the relative importance of thyroid volume and thyroid nodules as well as other possible factors influencing serum Tg. With the use of a sensitive immunoluminometric Tg-assay, special focus was on whether measuring serum Tg could be used to characterize the iodine status of a population in an epidemiological context. Subjects and Methods A population study, focusing on thyroid disease, was performed in urban areas in Copenhagen and Aalborg, 2 cities in Denmark. These locations were chosen because previous studies have shown that the western part of Denmark (Aalborg) suffers from moderate iodine deficiency and that the eastern part of Denmark (Copenhagen) suffers from mild iodine deficiency (19). The cohort comprised women in the age groups 18 22, 25 30, 40 45, and yr; and men, yr. From the Civil Registration System in Denmark, in which all citizens are registered with a unique 10-digit number, a sample comprising all inhabitants of the 2 areas in the selected ages was drawn. These 40,233 subjects were assigned random numbers, within each group, by a computer program. The subjects were then invited in order of the random numbers until the desired number of participants was reached in each group. The subjects were invited by letter; and in case of no response, a reminder was sent. Of the 9,274 subjects invited, 4,649 participated in the examination (50.1%). The cohort has previously been described in detail (20). The participants answered questionnaires concerning previous thyroid disease and medication, and further information about previous thyroid disease was obtained in an interview. Ultrasonography was performed with high-resolution ultrasonic equipment with a 7.5-MHz 70-mm linear transducer (Sonoline Versa Pro, Siemens, Erlangen, Germa- 3599

2 3600 The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8): Knudsen et al. The Epidemiology of Serum Thyroglobulin ny). Thyroid size was computed as length width depth of the each lobe /6, as previously described (21), and thyroid enlargement was defined as a thyroid vol exceeding 18 ml for women and 25 ml for men (22). The number of distinct nodules with a diameter exceeding 5 mm and the diameter of the nodules were recorded, and the structure of the glands was accordingly classified as regular, uninodular, or multinodular. These parameters have previously been shown to be reproducible (21). On the other hand, a heterogeneous echo pattern or hypoechogenecity was found not to be reproducible between different observers, and the association with serum Tg was not evaluated. Blood samples were drawn and stored at 20 C and analyzed after the completion of the investigation in a sequence mixing samples from the 2 regions, subjects of different age and gender, and samples collected at different seasons. Serum Tg and TSH were analyzed with immunoluminometric assays (LUMITEST, BRAHMS, Berlin, Germany) using a Stratec autoanalyzer (STRATEC-BIOMEDICAL SYSTEM AG, Birkenfeld, Germany). The Tg assay had an effective working range of g/liter (23). All assays included control samples at 3 levels at the start and end of each run. In 12 consecutive assays, the intraassay and interassay variations (coefficients of variation) for single determinations for controls with average Tg concentrations of 8.1, 45, and 154 g/liter were (intra) 5.3, 2.8, and 4.2% and (inter) 6.8, 4.5, and 3.3%. The assay was calibrated by the use of an in-house standardization procedure at BRAHMS; and for comparison to CRM457 calibrated assays, the values of LUMItest should be multiplied by 1.6 (manufacturer s information) (24). Tg Ab were measured with an RIA (DYNOtest, BRAHMS, Germany) with a functional assay sensitivity below 20 ku/ liter (manufacturer s information). In Table 1 it is seen that participants with Tg Ab levels above 20 ku/liter had an overrepresentation of very low values of serum Tg (22.4% of participants with serum Tg Ab 20 ku/liter had serum Tg 2 g/liter, compared with 3.3% of participants with Tg Ab 20 ku/liter). This illustrates that analytical interference was present and underlines the importance of exclusion of Tg Ab-positive samples from the Tg analyses. The functional sensitivity of the TSH assay was 0.01 mu/ liter, and the reference interval for serum TSH was set to mu/liter. For 4,554 participants (98.0%), serum Tg, Tg Ab, and TSH were all available. Spot urine samples were collected during the time interval 0800 to 1900 h and analyzed for iodine and creatinine concentration. For the iodine analysis, the urine samples were digested by alkaline ashing and analyzed for iodine content with the Ce/As method (25), as previously described (26). The functional sensitivity of the assay was 2 3 g/liter. Median urinary iodine concentration was 68 g/liter in Copenhagen and 53 g/liter in Aalborg; but after exclusion of subjects taking individual iodine supplementation, the figures were 61 g/liter in Copenhagen (mild iodine deficiency) and 45 g/liter in Aalborg (moderate iodine deficiency). Urinary creatinine concentration was analyzed with the kinetic method of Jaffe (27). Iodine excretion in the regression models was expressed as an age- and sex-adjusted iodine/creatinine ratio, because this has been shown to be an acceptable estimate of iodine excretion in individuals (28). Statistics All data processing was done with SPSS, Inc. version 8.0 software (SPSS Denmark, Holte, Denmark). In the linear models, factors were tested univariately and in a model including all significant factors. For analysis of continuous variables, Mann-Whitney tests and linear models were used. Because serum Tg was not normally distributed, but showed log normal distribution, logarithmic transformation was used before the linear model analyses. If interaction between terms was suspected, an interaction term was included; and in case of significant interactions, new variables, allowing analysis of the interacting terms as combined variables, were generated and included in the multivariate models. For the estimation of the strength of the association of the different factors to serum Tg, the type III Sums of Squares in the linear models were used, univariately as well as multivariately. Results Distribution of serum Tg The distribution of serum Tg values, stratified by region, is demonstrated in Table 1 with different selection criteria. When serum Tg was stratified by age and gender, marked regional differences were found in all groups as shown in Fig. 1. Factors associated with serum Tg Table 2 demonstrates the association between serum Tg and some demographic and physiological factors. Data are shown from a simple model where the association of the factors are demonstrated independently from each other, as well as from a multivariate model where adjustment is made for the impact TABLE 1. Serum Tg values in subjects from two areas in Denmark with slightly different degrees of ID Tg ( g/liter) mild ID Tg ( g/liter) moderate ID N a: All subjects with Tg results 10.9 ( ) 14.6 ( ) 4580 b: Tg antibodies 20 ku/liter 11.5 ( ) 15.4 ( ) 3928 c: b and no known thyroid disease 11.3 ( ) 15.2 ( ) 3759 d: c and no signs of thyroid disease a 9.5 ( ) 12.4 ( ) 2428 e: d and iodine supplementation b 7.4 ( ) 8.7 ( ) 571 Figures are medians, with 2.5 th and 97.5 th percentiles in brackets. Of the 4649 participants in the survey, Tg values were available in 4580 (98.5%). The difference between the regions was significant (P 0.001, Mann-Whitney) with all selection criteria. a No signs of thyroid disease including no thyroid enlargement and no nodules at ultrasonography and serum TSH in the range mu/liter. b Iodine supplementation, in nearly all cases, was taken as iodine containing (150 g) vitamin tablets.

3 Knudsen et al. The Epidemiology of Serum Thyroglobulin The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8): performed with stratification according to the regularity of the iodine intake and the daily dose, but no differences were detected between those groups. The impact of iodine supplementation on serum Tg was significantly stronger in the area with the most pronounced iodine deficiency (Fig. 2). FIG. 1. Serum Tgl in 3757 subjects randomly selected from the general population in 2 regions in Denmark with a different iodine intake. Subjects with known thyroid disease or Tg Ab more than 20 ku/liter were excluded. Values are means (after logarithmic transformation), and vertical bars represent 95% confidence intervals for means. The regional difference was significant in all groups; P 0.001; Mann-Whitney of the other factors. In the simple model, a significant association with serum Tg was found for all factors in the Table. The closest association was found for thyroid enlargement and thyroid nodularity, followed by iodine excretion and age- and sex-matched group. Significant associations were also found in the multivariate model for all factors, but the relative importance was different. Thyroid enlargement was still the most important factor, now followed by iodine excretion. The impact of thyroid nodules, and especially age and gender, diminished markedly in the multivariate model. Elevated serum TSH ( 5 mu/liter) and familial occurrence of thyroid disease had no significant impact on serum Tg in the multivariate model, and they are not shown. Thyroid volume (with r 0.49 and a regression coefficient of 1.0) had a close association with serum Tg levels (P 0.001). The association of serum Tg with serum TSH, when tested over the whole range of values, was not significant; and a correlation analysis between Tg and TSH showed an r of Iodine and serum Tg Living in areas with slightly different iodine status still had a significant impact on Tg levels (Table 2), even after adjusting for the measures of thyroid abnormalities and the estimates of iodine excretion. The estimated daily iodine excretion also had a marked association with serum Tg levels (Table 2). Decreasing levels of serum Tg were found with increasing iodine excretion,even with estimates of iodine excretion beyond the recommended minimum of g/d. A regular intake of iodine-containing vitamin tablets was reported by 1281 participants. In most cases, the daily supplementation was 150 g iodine, because this is the standard in Danish vitamin/mineral tablets; iodization of salt and other foods was not allowed in Denmark at this time. Analyses were Discussion In this comparative investigation of unselected subjects from two areas in Denmark, we report independent associations of thyroid volume, thyroid nodules, and thyroid dysfunction with levels of serum Tg. The most important predictor of serum Tg was clearly thyroid volume. Also, age and gender of the subjects had a significant relation to Tg levels, and marked regional differences in serum Tg were found in Denmark, probably conditioned by the different iodine status of the regions. The regional difference was still highly significant after adjusting for iodine excretion from casual urine samples, probably because iodine excretion in casual urine samples is a rather imprecise estimate of habitual iodine intake at the individual level (28). Previous studies have indicated that serum Tg is increased in patients with palpable goitre and in patients with palpable nodules in the thyroid (29 32). Contradictory reports have been published about the relation between thyroid volume at ultrasonography and serum Tg (33 35); this controversy might be caused by the limited number of participants in the studies or by the different iodine status of the populations. Previous studies have not been able to describe the independent influence of thyroid nodules and increased volume of the thyroid on serum Tg. We report independent and highly significant impacts on serum Tg of thyroid nodules and thyroid volume, suggesting that though the amount of thyroid tissue is important, also the structure of the thyroid tissue plays a role. It has been shown, in a number of studies, that serum Tg is increased in Graves disease (36, 37). We report an independent association of suppressed TSH values to serum Tg, though the associated increase in thyroid volume and nodules had additional effects. This is in contrast to Szabolcs et al. (31), who found no independent effect of suppressed TSH in a small study of geriatric patients. Controversy exists about the influence of age and gender on serum Tg, as discussed by Feldt-Rasmussen (36, 38, 39). We found clearly lower levels of serum Tg in men, also after correcting for the difference in thyroid abnormalities. This is in line with a report of increased Tg levels in women on estrogen treatment, suggesting an association to estrogen levels, though no gender difference was reported in that study (40). The effect of age was highly significant as a single factor; but adjusting for the age-related differences in the occurrence of thyroid abnormalities, the effect of age on serum Tg diminished considerably. This suggests that there may be little independent effect of age, but that the agerelated increase in serum Tg may simply be reflecting the increase in thyroid abnormalities. It is consistent with the reports of no age-related differences in serum Tg in iodinesufficient areas (39), where the prevalence of goitre is relatively constant with age. Since the report of a correlation between serum TSH and serum Tg in an endemic goitre area (41), the role of TSH in

4 3602 The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8): Knudsen et al. The Epidemiology of Serum Thyroglobulin TABLE 2. Demographic and thyroid-related factors associated to serum Tg levels Single factor Multivariate model Tg ( g/liter) P-value Tg ( g/liter) P-value Region Mild ID 10.8 Reference 20.2 Reference Moderate ID Group Women yr Women yr Women yr Women yr 17.3 Reference 25.3 Reference Men yr Iodine excretion 50 g/d g/d g/d g/d 10.2 Reference 19.3 Reference 250 g/d Thyroid enlargement Volume 18/25 mliter a 10.6 Reference 16.8 Reference Volume 18/25 mliter a Thyroid nodularity No nodules 10.4 Reference 17.3 Reference Single nodule Multinodular Hyperthyroidism TSH 0.2 mu/liter 12.6 Reference 16.5 Reference TSH 0.2 mu/liter Nodule size b 5 10 mm 14.2 Reference 16.3 Reference mm mm Data are from 3719 participants without known thyroid disease and with Tg antibodies 20 ku/liter. The analyses were done after logarithmic transformation of serum Tg; results are shown after transforming back to original values. In the single-factor model, only one of the factors were included, whereas in the multivariate model, adjustment was made for the impact of all other variables in the table. Multivariate values are given with an average contribution from the other factors. a Thyroid enlargement was defined as a volume exceeding 18 mliters for women and 25 mliters for men (22). b Only subjects with nodules 5 mm were included in this specific analysis. FIG. 2. Difference in the effect of iodine supplementation on serum Tgl levels in 2 areas with mild and moderate iodine deficiency. Data from 3757 Danes without known thyroid disease and with Tg Ab less than 20 ku/liter were included. Values are mean values, and vertical bars represent 95% confidence intervals for means; analyses were done after logarithmic transformation. P for the difference in the effect of iodine supplementation in the 2 regions (interaction in linear model). the control of Tg synthesis has been discussed. The correlation was confirmed in areas with severe iodine deficiency (ID) (17, 18), but reports from areas with less severe ID have failed to find any correlation between serum TSH and serum Tg (30, 38), and we found no correlation over the whole range of TSH in this study. A possible explanation is that, in severe iodine deficiency, goitre is associated to increased serum TSH values. This is not the case in areas with less severe iodine deficiency, such as ours, where goitre is rather associated to low serum TSH values, probably because of autonomous areas in the gland (42). Large differences in serum Tg were found in this study between the region with mild iodine deficiency and the region with moderate iodine deficiency, though the difference in iodine excretion was modest. The regional difference was also present after adjusting for differences in thyroid abnormalities and differences in iodine excretion. This suggests that Tg may be a sensitive and useful marker of iodine deficiency if standardized Tg assays are used. Other studies investigating regional differences in Tg in areas with more pronounced differences in iodine excretion have found large differences in serum Tg (12, 13, 18). One study indicated that abundant iodine intake may also lead to increased Tg levels, though the difference was insignificant in the small material (14). Such a tendency was not found within the range of iodine excretion in this study; serum Tg decreased with increasing iodine excretion over the whole range of iodine excretion. The large impact of iodine supplementation on serum Tg in the area with moderate ID emphasizes the sensitivity of serum Tg as a marker of iodine deficiency. The small influence of iodine supplementation in the region with only mild iodine deficiency probably reflects Tg values closer to the optimal levels in iodine sufficiency. Thus, only little further reduction could be obtained with iodine supplementation. This was parallel to experimental data, where no effect of iodine supplementation to iodine-replete rats was seen, whereas iodine deficient rats had increased serum Tg (43). The different impact on serum Tg of iodine supplementation

5 Knudsen et al. The Epidemiology of Serum Thyroglobulin The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8): in the two regions also underlines the difference in thyroid physiology and pathology between areas with mild and moderate iodine deficiency. In conclusion, serum Tg is a marker of thyroid abnormalities and thyroid dysfunction, with thyroid volume as the most important determinant. In population studies, Tg is a sensitive marker of iodine deficiency (in many situations, easier to apply than thyroid volume measurement or measurement of iodine excretion). Especially with the newer assays with better standardization, higher sensitivity, less problems with interference from Tg Ab, and increasing automation, serum Tg may be a useful tool in the monitoring of iodine status in a population. Acknowledgments We express our thanks to Inge-Lise Legaard and Rene Fiege, who carefully performed the ultrasonographies; and to BRAHMS, who provided the kits for the biochemical analyses at very favorable conditions. Received January 10, Accepted April 27, Address all correspondence and requests for reprints to: Nils Knudsen, Medical Clinic I, Bispebjerg Hospital, DK-2400 Copenhagen NV, Denmark. danthyr.bbh@dadlnet.dk. This study was part of The Danish Investigation of Iodine Intake and Thyroid diseases, which was supported by grants from Tømmerhandler Vilhelm Bang Foundation, the Copenhagen Hospital Corporation Research Foundation, the 1991 Pharmacy Foundation, the Danish Medical Foundation, the Health Insurance Foundation, and North Jutland County Research Foundation. References 1. Van-Herle AJ, Uller RP 1975 Elevated serum thyroglobulin. A marker of metastases in differentiated thyroid carcinomas. 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