Association between serum thyroid hormone balance and thyroid volume in patients treated with levothyroxine monotherapy for hypothyroidism

Abstract

Many previous studies including ours have reported that athyreotic patients on levothyroxine (LT₄) have relatively low serum free triiodothyronine (FT₃) levels, whereas patients with large goitrous diseases often have high serum FT₃ levels. Here we investigated Hashimoto thyroiditis (HT) patients on LT₄ to study the relationship between thyroid volume (TV) and thyroid hormone status in hypothyroid patients on LT₄. We retrospectively studied 408 euthyroid HT patients treated with LT₄ for hypothyroidism; divided them as per TV and compared serum levels of free thyroxine (FT₄) and FT₃ and the FT₃/FT₄ ratio in each patient group with those in euthyroid matched control group. We also evaluated the association between serum FT₃ level and FT₃/FT₄ ratio and TV among HT patients on LT₄. In patients with TV <15 mL, serum FT₃ levels were significantly lower than those in controls. In patients with TV 15–80 mL, serum FT₃ levels were equivalent to those in controls. In patients with TV ≥80 mL, the serum FT₃ levels were significantly higher than those in controls. The serum FT₃ level (r = 0.35, p < 0.01) and FT₃/FT₄ ratio (r = 0.42, p < 0.01) showed a positive correlation with TV. TVs in HT patients on LT₄ caused differences in serum thyroid hormone balance, as increasing volume increases the serum FT₃ level and FT₃/FT₄ ratio. Serum thyroid hormone balance in HT patients with smaller thyroids was similar to that in athyreotic patients. Mild thyrotropin suppression with LT₄ is needed to achieve normal FT₃ levels in such patients.

THE TWO MAJOR THYROID HORMONES in the body are triiodothyronine (T₃) and thyroxine (T₄). Approximately 20% of T₃ is produced from the thyroid gland via two pathways, as follows: coupling of monoiodotyrosine and diiodotyrosine (DIT) and conversion of T₄ to T₃ by type 1 and type 2 iodothyronine deiodinases (D1 and D2, respectively). The remaining 80% of T₃ is derived from the conversion of T₄ to T₃ in extrathyroidal tissues. In contrast, 100% of T₄ is secreted by the thyroid gland through the coupling of two DIT moieties [1].

Some previous studies including ours have reported normal serum thyrotropin (TSH) levels associated with mildly low serum free triiodothyronine (FT₃) levels in patients on levothyroxine (LT₄) monotherapy for athyreotic or atrophic conditions after total thyroidectomy or after radioiodine treatment for Graves’ disease [2-5]. In addition, we have reported that the presence of the remnant thyroid tissue was associated with normal FT₃ levels in patients treated with LT₄ who underwent hemithyroidectomy [6] or radioiodine treatment for Graves’ disease [5]. In contrast, we documented that Hashimoto thyroiditis (HT) patients with increased thyroid volume (TV) tended to present with high serum FT₃ levels, low free thyroxine (FT₄) levels, and high FT₃/FT₄ ratios [7, 8]. Thus, TV may be an important factor affecting thyroid hormonal balance, including serum FT₃ levels and FT₃/FT₄ ratios.

In the present study, we investigated the thyroid hormone balance among HT patients during LT₄ monotherapy for hypothyroidism who presented with a variety of TVs, and we elucidated the relationship between TV and thyroid hormone status in hypothyroid patients on LT₄.

Materials and Methods

Patients

We retrospectively identified 408 consecutive patients (379 women and 29 men) with HT from hospital medical records who visited the Kuma Hospital between January 2012 and May 2018. We based the diagnosis of HT on the presence of anti-thyroglobulin antibody (TgAb) positivity and/or anti-thyroid peroxidase antibody (TPOAb) positivity, and a heterogeneous hypoechoic pattern in a thyroid ultrasound examination.

The inclusion criteria were as follows: (1) underwent an ultrasound examination and TV was measured, (2) administered LT₄ before a thyroid ultrasound examination, and (3) TSH level within the laboratory reference range (0.3–5.0 μIU/mL) on a thyroid ultrasound examination. The exclusion criteria were as follows: (1) follicular adenoma and thyroid malignancies, (2) thyroid dysfunction, such as Graves’ disease, thyroid dyshormonogenesis, autonomously functioning thyroid nodules, or hypothyroidism, (3) administered drugs known to affect thyroid function or thyroid hormone metabolism, such as a steroids, estrogen, amiodarone, lithium, β-blockers, sucralfate, and iron or iodine-containing drugs, (4) chronic or serious diseases, such as cardiac, pulmonary, hepatic, renal, or pancreatic diseases, diabetes or hyperparathyroidism, and (5) pregnant or lactating women. In addition to the above exclusion criteria, patients who failed to achieve the target TSH levels were also excluded from the analysis.

Control subjects

Overall, 1,149 consecutive euthyroid subjects (901 women and 248 men) who were examined for possible thyroid abnormalities at the Kuma Hospital during the same period as that of patients and did not have clinical or laboratory signs of thyroid diseases served as controls. Subjects with positive TPOAb or TgAb test results or with abnormal findings on ultrasound examination were excluded. Subjects with a thyroidal nodule or a goiter (TV: men ≥20 mL and women ≥18 mL) [9] on an ultrasound examination were also excluded. The other exclusion criteria were the same as those used for the selection of the patients. We balanced covariates including age, sex, and the measured year for choosing the control subjects for each patient group. Control subjects for each group of patients were chosen from 1,149 subjects selected earlier by 1:1 matching. This study was approved by the Ethical Committee at Kuma Hospital, and all patients gave written, informed consent.

Laboratory serum tests

For the patients who were taking LT₄, blood samples were obtained in the morning after the ingestion of LT₄. The patients’ serum levels of TSH, FT₄, and FT₃ were measured using a chemiluminescent immunoassay (ARCHTECT i2000; Abbott Japan, Tokyo). The intra-assay coefficients of variation and the inter-assay coefficients of variation were 1.1%–5.0% and 1.7%–5.3% for the TSH assay, 2.3%–5.3% and 3.6%–7.8% for the FT₄ assay, and 1.4%–4.2% and 2.3%–5.0% for the FT₃ assay, respectively. The reference ranges in our hospital are 0.3–5.0 μIU/mL for TSH, 0.7–1.6 ng/dL for FT₄, and 1.7–3.7 pg/mL for FT₃. The serum levels of TgAb and TPOAb were measured using an electrochemiluminescence immunoassay (ECLusys 2010; Roche Diagnostics Japan, Tokyo; normal range: <40 IU/mL for TgAb, <16 IU/mL for TPOAb). A TgAb level less than 40 IU/mL was regarded as 40 IU/mL and that more than 4,000 IU/mL was regarded as 4,000 IU/mL, for the purpose of statistical calculations. A TPOAb level less than 16 IU/mL was regarded as 16 IU/mL and that more than 600 IU/mL was regarded as 600 IU/mL, for the purpose of statistical calculations. TV was measured using an ultrasound, as reported previously. First, the maximum width (W), maximum thickness (T), and maximum length (L) were measured in the right lobe (r) and left lobe (l). Second, TV was calculated by the following equation: TV = 0.70 (Wr × Tr × Lr + Wl × Tl × Ll) [10].

Statistical analysis

Grouped data are expressed as the mean ± standard deviation or the median (25^th to 75^th percentiles). Group comparisons among the HT patients stratified according to TV were analyzed using the χ² test (sex), Tukey-Kramer test, or Steel-Dwass. Treatment effects (control vs. HT patients on LT₄ for hypothyroidism) were analyzed using the paired t-test for data with a normal distribution and the Wilcoxon signed rank test for data with a nonparametric distribution. Significance was defined as p-value <0.05 (two-sided).

Results

Characteristics among the HT patient groups stratified according to TV

Baseline characteristics data of the HT patients are listed in the Table 1. In the present study, all patients had normal serum TSH levels. We stratified the patients into seven groups according to their TVs, as follows: <5 mL, 5–10 mL, 10–15 mL, 15–20 mL, 20–50 mL, 50–80 mL, and ≥80 mL. In the context of patients by TV, LT₄ doses tended to be higher in the atrophy group. There was an association between serum FT₃/FT₄ level and LT₄ dose in each group with 5 mL ≤TV. In contrast, there was no correlation between FT₃ level and LT₄ dose (data were not shown).

Table 1 Baseline characteristics sub grouped by thyroid volume in patients with Hashimoto’s thyroiditis

Patient subgroups: (mL)	G1 TV <5	G2 5 ≤ TV < 10	G3 10 ≤ TV < 15	G4 15 ≤ TV < 20	G5 20 ≤ TV < 50	G6 50 ≤ TV < 80	G7 TV ≥80
n (male)	25 (1)	48 (7)	53 (4)	57 (5)	96 (6)	50 (1)	79 (5)
Age (years)	64 ± 11	62 ± 15	54 ± 17	52 ± 17	58 ± 14	61 ± 13	66 ± 10c,d
LT4 dose (μg/day)	94 ± 19	80 ± 29	63 ± 30a	33 ± 62a	67 ± 28a	57 ± 27a	64 ± 29a
TSH (μIU/mL)	1.10 (0.62–1.62)	1.32 (0.78–2.52)	2.24a (1.32–3.15)	1.55 (0.90–2.70)	1.98 (1.05–3.28)	1.99a (1.38–3.67)	1.21c,e,f (0.63–1.90)
FT4 (ng/dL)	1.16 (1.13–1.23)	1.16 (1.08–1.25)	1.08 (1.02–1.20)	1.10 (1.02–1.20)	1.06 (0.95–1.21)	0.96a,b,c,d (0.87–1.11)	1.04a,b,d (0.89–1.14)
FT3 (pg/mL)	2.59 (2.36–2.79)	2.62 (2.43–2.84)	2.64 (2.45–2.84)	2.68 (2.48–2.93)	2.73 (2.50–2.93)	2.82a,b (2.57–3.00)	2.92a,b,c,d,e (2.74–3.16)
TgAb (IU/mL)	345.0 (93.7–2,962.0)	238.4 (115.1–592.3)	345.6 (123.6–672.8)	502.5 (322.9–1,026.0)	423.5 (217.7–788.5)	418.0 (272.7–561.5)	509.3 (385.2–744.7)
TPOAb (IU/mL)	40.9 (16.0–164.4)	64.3 (16.0–226.0)	183.9 (34.4–441.1)	160.4 (29.0–354.3)	375.6a,b (104.3–600.0)	337.5a,b (40.9–600.0)	214.9a (20.7–600)

Abbreviations: TV, Thyroid volume; TSH, thyroid stimulating hormone; FT₄, free thyroxine; FT₃, free triiodothyronine.

Values shown are the means ± SD in case of normal distribution and the medians (25–75% tile) in case of nonparametric distribution. Statistical significance was analyzed by the χ² test (sex), Tukey-Kramer test) or Steel-Dwass test for multiple comparisons.

^a p < 0.05, compared with G1, ^b p < 0.05, compared with G2, ^c p < 0.05, compared with G3, ^d p < 0.05, compared with G4, ^e p < 0.05, compared with G5, ^f p < 0.05, compared with G6.

Serum thyroid hormone levels in HT patients stratified according to TV and those in the matched euthyroid controls

In patients with TV levels <5 mL, 5–10 mL, and 10–15 mL, the serum FT₃ levels were significantly lower than those in the matched controls (p < 0.05, p < 0.001, p < 0.05, respectively). In patients with TV levels 15–20 mL, 20–50 mL, and 50–80 mL, the serum FT₃ levels were equivalent to those in the matched controls (p = 0.109, p = 0.111, p = 0.452, respectively). In patients with TV levels ≥80 mL, the serum FT₃ levels were significantly higher than those in the matched controls (p < 0.05) (Fig. 1A).

Fig. 1

Serum levels of FT₃ (A), FT₄ (B) and FT₃/FT₄ (C) in patients with Hashimoto thyroiditis (HT) and in euthyroid controls with intact thyroid matched by age, sex, and the measured year. The HT patients were divided into seven groups stratified by TV levels. The top, bottom, and middle lines of the boxes correspond to the 75^th, 25^th, and 50^th percentiles (median), respectively. The whiskers extend from the minimum to the maximum. TV, Thyroid volume; FT₃, free triiodothyronine; FT₄, free thyroxine; TSH, thyrotropin.

In patients with TV levels <5 mL, 5–10 mL, and 10–15 mL, the serum FT₄ levels were significantly higher than those in the matched controls (p < 0.05, p < 0.001, p < 0.05, respectively). In patients with TV levels 15–20 mL, 20–50 mL, 50–80 mL, and ≥80 mL, the serum FT₄ levels were equivalent to those in matched controls (p = 0.157, p = 0.158, p = 0.251, p = 0.844, respectively) (Fig. 1B).

In patients with TV levels <5 mL, 5–10 mL, and 10–15 mL, the serum FT₃/FT₄ ratios were significantly lower than those in the matched controls (p < 0.001, p < 0.001, p < 0.001, respectively). In patients with TV levels 15–20 mL, 20–50 mL, and 50–80 mL, the serum FT₃/FT₄ ratios were equivalent to those in the matched controls (p = 0.077, p = 0.055, p = 0.094, respectively). In patients with TV levels ≥80 mL, the serum FT₃/FT₄ ratios were significantly higher than those in the matched controls (p < 0.05) (Fig. 1C).

Association between serum FT₃ levels and FT₃/FT₄ ratios and TV in HT patients treated with LT₄ for hypothyroidism

The correlations between serum FT₃ levels and FT₃/FT₄ ratios and TV in HT patients treated with LT₄ for hypothyroidism were evaluated. The serum FT₃ levels showed a positive correlation with TV (r = 0.35, p < 0.01; Fig. 2A). The serum FT₃/FT₄ ratios also showed a positive correlation with TV (r = 0.42, p < 0.01; Fig. 2B).

Fig. 2

(A) Association between serum free triiodothyronine and thyroid volume using the Pearson’s correlation coefficient test among Hashimoto thyroiditis patients. (B) Association between serum free triiodothyronine/free thyroxine ratio and thyroid volume using the Pearson’s correlation coefficient test among Hashimoto thyroiditis patients.

Discussion

In the present study, in patients with small or normal TVs (<15 mL), the serum FT₃ levels significantly lower than those of controls. Thus, these data were similar to those from previous studies of athyreotic patients on LT₄ who underwent total thyroidectomy [2] or atrophic thyroid patients on LT₄ who underwent radioiodine treatment for Graves’ disease [5]. In contrast, in patients with slight or moderate goiter (TV, 20–80 mL), serum FT₃ levels were equivalent to those of the controls. These data were consistent with those from previous studies of patients on LT₄ who underwent a hemithyroidectomy [6] or of a certain volume of thyroid gland (TV >10 mL) patients on LT₄ who underwent radioiodine treatment for Graves’ disease [5]. These findings suggest that the reason underlying the decreased serum T₃ levels in such patients is the lack of intra-thyroidal T₃ production caused by atrophy or loss of the thyroid gland.

There are two types of deiodinases (D1 and D2) that contribute to T₃ production. The serum FT₃/FT₄ ratio reflects the activity of the deiodinase enzyme, which converts T₄ to T₃ by 5'-deiodination [11]. Maia et al. estimated that D2 is the major contributor of extrathyroidal T₃ production in euthyroid subjects [12]. While, Hoermann et al. indicated that LT₄-treated patients with a post-interventional lower residual volume (<5 mL) have significantly reduced deiodinase activity and lowered T₃ levels, as compared with patients with a higher residual TV [13]. Such atrophic or athyreotic patients have reduced thyroidal deiodinase activity and T₃ production from the thyroid gland, resulting in a relatively low serum FT₃ level and FT₃/FT₄ ratio [6]. It is necessary to clarify whether D1 and/or D2 activity in the thyroid tissues of such patients contributes to the patients’ lower serum FT₃ level and serum FT₃/FT₄ ratios.

Our present study revealed that HT patients with large goiter (TV ≥80 mL) had relatively high serum FT₃ levels and a high FT₃/FT₄ ratio. We also demonstrated a positive correlation between the serum FT₃/FT₄ ratio and TV, findings consistent with those from our previous report [7, 8]. Elevation of thyroidal deiodinase activity, predominantly that of D2 activity, was reported as the cause of serum FT₃ level and FT₃/FT₄ ratio elevation in several large goitrous thyroid diseases, such as those involving thyroglobulin gene mutations, McCune-Albright syndrome, and T₃-predominant Graves’ disease [14-16]. Recently, we reported the elevation of thyroidal deiodinase activity (especially D2 activity) at the posttranslational level in seven HT patients with large goiters; this increase in enzyme activity may be responsible for the relatively high serum FT₃/FT₄ ratio observed in these patients [7]. We also demonstrated a positive correlation between deiodinase activities and TV [8]. These findings suggest that intra-thyroidal T₃ production by increased deiodinase activity is a substantial factor influencing the relatively high serum T₃ levels in HT patients with large goiters. These may also be related to higher LT₄ doses when the goiter was small and lower LT₄ doses when the goiter was large in the present study. These results suggest that thyroid tissue capacity plays a significant role in the physiological process of T₃ homeostasis in humans; this contention fits well with the results from the present study.

In atrophic or normal thyroid size patients on LT₄, patients with normal TSH levels had relatively low serum FT₃ levels. The question arises as to whether such a patient is in a euthyroid condition. In the athyreotic patients with normal TSH and low T₃ levels, the relatively higher serum T₄ levels that accompany LT₄ monotherapy seems to result in normal T₃ receptor occupancy and TSH in pituitary thyrotrophs. In contrast, in peripheral tissues, the relatively higher serum T₄ levels could impair intracellular T₃ production via downregulation of a D2 pathway [17]. In fact, an animal study has shown that LT₄ alone administered in thyroidectomized rats at doses to normalize plasma TSH levels does not normalize T₃ contents in some tissues [18]. In another study of rats, Werneck et al. reported that a combination of high serum T₄ and low serum T₃ levels during T₄ monotherapy in rats had consequences of thyroid hormone action, as reflected in the brain, liver, and skeletal muscle, all of which exhibited indications of hypothyroidism despite normal serum TSH level [17]. In a previous study conducted in humans, we compared biochemical markers reflecting thyroid function before and after thyroidectomy. Therefore, the biochemical markers suggest that the patients with mildly suppressed TSH levels were closest to euthyroid, whereas those with normal TSH levels were mildly hypothyroid [19]. Recently, in a large LT₄-treated population with normal serum TSH, participants exhibited lower serum T₃ levels and differed in terms of both objective and subjective measures [20]. In addition, a meta-analysis performed by McAninch et al. showed that serum total cholesterol and low-density lipoprotein levels remain high in LT₄-treated euthyroid patients in meta-analysis [21].

The symptoms of thyroidal dysfunction have also been reported by several prior studies. Recently, we compared reported subjective symptoms reflecting thyroid function before and after thyroidectomy. Therefore, the symptoms suggest that the patients with mildly suppressed TSH levels were closest to euthyroid, whereas those with normal TSH levels were mildly hypothyroid [22]. Larisch et al. conducted a retrospective longitudinal study including patients with differentiated thyroid carcinoma on LT₄. Therefore, 26% of patients expressed hypothyroid and 9.7% hyperthyroid complaints at any one visit. Hypothyroid symptoms correlated well with FT₃ levels and were observed when TSH levels were below the reference range [23]. The American Thyroid Association stated in its guidelines that for the treatment of hypothyroidism such as in athyreotic patients, there is insufficient evidence of benefit to recommend LT₄ treatment for achieving low-normal TSH values or high-normal T₃ values [24]. Overall, the presence of biochemical markers and symptoms of thyroid function in animal and human studies of LT₄-treated athyreotic conditions suggests that patients with normal TSH might not be euthyroid in all tissues, and mild TSH suppression with LT₄ might be needed to achieve euthyroidism.

In the present study, the patients on LT₄ with large goitrous thyroid had relatively high serum FT₃ levels. It is unclear currently whether relatively high serum FT₃ in such patients presents with thyrotoxicosis. The clinical significance of the relative high serum FT₃ levels in patients with large goiter should be evaluated in the future.

There are some limitations in the present study. First, the limited number of study patients, unequal group distribution, and single time point reduced the internal validity of the study. Second, we did not evaluate biochemical markers and the symptoms reflecting thyroid function. In addition, we did not evaluate echo pattern (low or normal) of HT patients. Studies including measures of these clinical parameters are thus needed to clarify the best method of managing HT patients’ thyroid function on LT₄.

LT₄ has been considered the standard of care for treatment of hypothyroidism for many years. LT₄ replacement therapy has three main goals: (i) provide resolution of the patients’ symptoms and hypothyroid signs, including biological and physiologic markers of hypothyroidism, (ii) achieve normalization of serum TSH with improvement in thyroid hormone concentrations, and (iii) avoid overtreatment [24]. This study suggests that one of the three main goals has a pitfall. Patients with hypothyroidism due to HT are treated with LT₄ and live in a chronic condition of abnormal thyroid hormone status for their lives. Therefore, even if the thyroidal dysfunction may be subtle, its long-term effects cannot be overlooked. We analyzed a large number of HT patients on LT₄ and demonstrated that their TVs caused differences in serum thyroid hormone balance. The patients with normal TSH levels had relatively high serum FT₃ levels and FT₃/FT₄ ratios as their goiter size increased. Thus, serum low FT₃ levels in HT patients on LT₄ with relatively small thyroid were consistent with those in athyreotic patients on LT₄, and serum high FT₃ levels in HT patients on LT₄ with relatively large thyroid were consistent with those in patients with large goitrous thyroid disease. In the former, the possibility of mild hypothyroidism has been suggested, and mild TSH suppression with LT₄ may be needed to achieve normal FT₃ levels. The clinical significance of the latter is for further study. Our findings may provide novel information that could assist in the management of a large number of patients treated with LT₄ for hypothyroidism.

Acknowledgments

Author contributions: M. Ito constructed the study design. S. Takahashi analyzed the data. The other coauthors contributed by administering patient care.

Disclosure Statement

The authors declare no competing financial interests.

References

• 1   Pilo  A,  Iervasi  G,  Vitek  F,  Ferdeghini  M,  Cazzuola  F, et al. (1990) Thyroidal and peripheral production of 3,5,3'-triiodothyronine in humans by multicompartmental analysis. Am J Physiol 258: E715–E726.
• 2   Ito  M,  Miyauchi  A,  Morita  S,  Kudo  T,  Nishihara  E, et al. (2012) TSH-suppressive doses of levothyroxine are required to achieve preoperative native serum triiodothyronine levels in patients who have undergone total thyroidectomy. Eur J Endocrinol 167: 373–378.
• 3   Gullo  D,  Latina  A,  Frasca  F,  Le Moli  R,  Pellegriti  G, et al. (2011) Levothyroxine monotherapy cannot guarantee euthyroidism in all athyreotic patients. PLoS One 6: e22552.
• 4   Hoermann  R,  Midgley  JE,  Giacobino  A,  Eckl  WA,  Wahl  HG, et al. (2014) Homeostatic equilibria between free thyroid hormones and pituitary thyrotropin are modulated by various influences including age, body mass index and treatment. Clin Endocrinol (Oxf) 81: 907–915.
• 5   Ito  M,  Kawasaki  M,  Danno  H,  Kohsaka  K,  Nakamura  T, et al. (2019) Serum thyroid hormone balance in levothyroxine monotherapy-treated patients with atrophic thyroid after radioiodine treatment for Graves’ disease. Thyroid 29: 1364–1370.
• 6   Ito  M,  Miyauchi  A,  Kang  S,  Hisakado  M,  Yoshioka  W, et al. (2015) Effect of the presence of remnant thyroid tissue on the serum thyroid hormone balance in thyroidectomized patients. Eur J Endocrinol 173: 333–340.
• 7   Harada  A,  Nomura  E,  Nishimura  K,  Ito  M,  Yoshida  H, et al. (2019) Type 1 and type 2 iodothyronine deiodinases in the thyroid gland of patients with huge goitrous Hashimoto’s thyroiditis. Endocrine 64: 584–590.
• 8   Kawasaki  M,  Ito  M,  Danno  H,  Kousaka  K,  Nakamura  T, et al. (2019) The association between thyroid hormone balance and thyroid volume in patients with Hashimoto thyroiditis. Endocr J 66: 763–768.
• 9   Kitaoka  M,  Suzuki  S (ed) (2016) Thyroid Ultrasound – A guidebook for diagnosis and management (3rd edition). Japan Association of Breast and Thyroid Sonology. Nankodo, Tokyo, Japan (In Japanese).
• 10   Murakami  Y,  Takamatsu  J,  Sakane  S,  Kuma  K,  Ohsawa  N (1996) Changes in thyroid volume in response to radioactive iodine for Graves’ hyperthyroidism correlated with activity of thyroid-stimulating antibody and treatment outcome. J Clin Endocrinol Metab 81: 3257–3260.
• 11   Bianco  AC,  Salvatore  D,  Gereben  B,  Berry  MJ,  Larsen  PR (2002) Biochemistry, cellular and molecular biology, and physiological roles of the iodothyronine selenodeiodinases. Endocr Rev 23: 38–89.
• 12   Maia  AL,  Kim  BW,  Huang  SA,  Harney  JW,  Larsen  PR (2005) Type 2 iodothyronine deiodinase is the major source of plasma T3 in euthyroid humans. J Clin Invest 115: 2524–2533.
• 13   Hoermann  R,  Midgley  JE,  Larisch  R,  Dietrich  JW (2015) Integration of peripheral and glandular regulation of triiodothyronine production by thyrotropin in untreated and thyroxine-treated subjects. Horm Metab Res 47: 674–680.
• 14   Kanou  Y,  Hishinuma  A,  Tsunekawa  K,  Seki  K,  Mizuno  Y, et al. (2007) Thyroglobulin gene mutations producing defective intracellular transport of thyroglobulin are associated with increased thyroidal type 2 iodothyronine deiodinase activity. J Clin Endocrinol Metab 92: 1451–1457.
• 15   Celi  FS,  Coppotelli  G,  Chidakel  A,  Kelly  M,  Brillante  BA, et al. (2008) The role of type 1 and type 2 5'-deiodinase in the pathophysiology of the 3,5,3'-triiodothyronine toxicosis of McCune-Albright syndrome. J Clin Endocrinol Metab 93: 2383–2389.
• 16   Ito  M,  Toyoda  N,  Nomura  E,  Takamura  Y,  Amino  N, et al. (2011) Type 1 and type 2 iodothyronine deiodinases in the thyroid gland of patients with 3,5,3'-triiodothyronine-predominant Graves’ disease. Eur J Endocrinol 164: 95–100.
• 17   Werneck de Castro  JP,  Fonseca  TL,  Ueta  CB,  McAninch  EA,  Abdalla  S, et al. (2015) Differences in hypothalamic type 2 deiodinase ubiquitination explain localized sensitivity to thyroxine. J Clin Invest 125: 769–781.
• 18   Escobar-Morreale  HF,  Obregón  MJ,  Escobar del Rey  F,  Morreale de Escobar  G (1995) Replacement therapy for hypothyroidism with thyroxine alone does not ensure euthyroidism in all tissues, as studied in thyroidectomized rats. J Clin Invest 96: 2828–2838.
• 19   Ito  M,  Miyauchi  A,  Hisakado  M,  Yoshioka  W,  Ide  A, et al. (2017) Biochemical markers reflecting thyroid function in athyreotic patients on levothyroxine monotherapy. Thyroid 27: 484–490.
• 20   Peterson  SJ,  McAninch  EA,  Bianco  AC (2016) Is a normal TSH synonymous with “euthyroidism” in levothyroxine monotherapy? J Clin Endocrinol Metab 101: 4964–4973.
• 21   McAninch  EA,  Rajan  KB,  Miller  CH,  Bianco  AC (2018) Systemic thyroid hormone status during levothyroxine therapy in hypothyroidism: a systematic review and meta-analysis. J Clin Endocrinol Metab 103: 4533–4542.
• 22   Ito  M,  Miyauchi  A,  Hisakado  M,  Yoshioka  W,  Kudo  T, et al. (2019) Thyroid function related symptoms during levothyroxine monotherapy in athyreotic patients. Endocr J 66: 953–960.
• 23   Larisch  R,  Midgley  JEM,  Dietrich  JW,  Hoermann  R (2018) Symptomatic relief is related to serum free triiodothyronine concentrations during follow-up in levothyroxine-treated patients with differentiated thyroid cancer. Exp Clin Endocrinol Diabetes 126: 546–552.
• 24   Jonklaas  J,  Bianco  AC,  Bauer  AJ,  Burman  KD,  Cappola  AR, et al. (2014) Guidelines for the treatment of hypothyroidism. Thyroid 24: 1670–1751.