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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2006-2217
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 8 3108-3113
Copyright © 2007 by The Endocrine Society

Evidence for Thyroid Hormone as a Positive Regulator of Serum Thyrotropin Bioactivity

Juliana H. A. Oliveira, Erika R. Barbosa, Teresa Kasamatsu and Julio Abucham

Neuroendocrine Unit, Endocrine Division, Universidade Federal de São Paulo, São Paulo, SP 04039-002, Brazil

Address all correspondence and requests for reprints to: Julio Abucham, M.D., Ph.D., Neuroendocrine Unit, Endocrinology Division, Department of Medicine, Universidade Federal de São Paulo, Rua Pedro de Toledo, 910. São Paulo 04039-002, Brazil. E-mail: julioabucham{at}nw.com.br.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: The regulation of TSH bioactivity in humans is not completely understood.

Objective: The aim of the study was to investigate the role of serum thyroid hormones in regulating the bioactivity of TSH.

Design: We determined in vitro TSH bioactivity and glycosylation in nine patients (six females and three males, age 41.3 yr) with primary hypothyroidism before and after L-T4 replacement, in 11 age- and sex-comparable controls (seven females and four males, age 37.6 yr), and in two thyroidectomized patients with TSH-secreting adenomas during and after L-T4 withdrawal.

Methods: In vitro TSH bioactivity was measured by a sensitive and specific bioassay based on cAMP generation by Chinese hamster ovary cells transfected with human TSH receptor. TSH glycosylation was assessed by concanavalin A lectin and ricin column affinity chromatography.

Results: In vitro TSH bioactivity in hypothyroid patients was low as compared with controls (0.48 ± 0.1 vs. 1.1 ± 0.2; P = 0.004) and increased during L-T4 (0.48 ± 0.1 vs. 0.8 ± 0.1; P = 0.01). A strong significant correlation (r = +0.80; P = 0.004, Spearman) was observed between the absolute increments of serum TSH bioactivity and T3 during L-T4 replacement. The degree of sialylation was elevated in hypothyroid patients before treatment (47 ± 2.4% vs. 29 ± 4.3%; P = 0.002) and decreased significantly after L-T4 (47 ± 2.4% vs. 33 ± 4.3%; P = 0.02). The mannose content of serum TSH in hypothyroid patients was similar to controls and did not change during L-T4. In vitro TSH bioactivity also decreased in patients with TSH-secreting adenomas during L-T4 withdrawal.

Conclusion: These data indicate that serum thyroid hormone level is a positive regulator of TSH bioactivity.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
TSH IS A PITUITARY glycoprotein hormone composed of two noncovalently linked {alpha}- and ß-peptide subunits. Both TSH subunits are cotranslationally glycosylated in specific asparagine residues with precursor oligosaccharides rich in mannose. Posttranslationally, these subunits combine, and further processing of the oligosaccharides occurs in the rough endoplasmic reticulum and Golgi apparatus. The resulting mature TSH molecules possess complex bi- and tri-antennary carbohydrate structures with decreased mannose content and a terminal sulfate or sialic acid cap (1, 2, 3). Circulating TSH has multiple molecular isoforms with variable carbohydrate structure and biological activity (4, 5).

The regulation of TSH bioactivity in humans is not completely understood. TRH has been considered a major positive regulator of TSH bioactivity because chronic administration of TRH to patients with hypothalamic hypothyroidism increases in vitro serum TSH bioactivity and thyroid hormone levels (6). More recently, we have shown that patients with pituitary hypothyroidism due to postpartum pituitary necrosis (Sheehan’s syndrome), a condition that does not affect the hypothalamus, also present TSH abnormalities similar to patients with hypothalamic hypothyroidism, with normal or slightly increased serum TSH levels with decreased biological activity (7, 8, 9).

To investigate the relative role of serum thyroid hormones in regulating the biological activity of circulating TSH, we determined in vitro serum TSH bioactivity and glycosylation in patients with primary hypothyroidism (a "low thyroid hormone/high TRH" condition) before and after L-T4 replacement therapy (a "normal thyroid hormone/normal TRH" condition). In vitro serum TSH bioactivity was also determined in two thyroidectomized patients with TSH-secreting adenomas during high-dose L-T4 replacement (a "low TRH/high thyroid hormone" condition) and after short-term L-T4 withdrawal. Because the few reported studies of in vitro serum TSH bioactivity in primary hypothyroidism have shown conflicting results (10, 11, 12, 13), we sought to revisit this issue with improved study design, using the same group of patients before and after L-T4 treatment, an age and sex comparable control group, and a last generation TSH bioassay with improved sensitivity.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients and controls

Nine patients with untreated primary hypothyroidism (three men and six women, age 41.3 ± 2.4 yr, range 33–54), 11 age and sex comparable normal control subjects (four men and seven women, age 37.6 ± 2.4 yr, range 28–52), and two male patients (36 and 47 yr) on supraphysiological L-T4 replacement after incomplete resection of TSH-secreting pituitary adenomas and surgical/radioiodine thyroid ablation were studied. Informed consent was obtained from all patients and controls. Approval of the study protocol was obtained from the ethical committee of Hospital São Paulo-Universidade Federal de São Paulo. The diagnosis of primary hypothyroidism was established by high-serum TSH and low-serum free T4 (FT4) levels. Hypothyroidism was due to thyroidectomy (n = 2), radioiodine therapy (n = 4), and Hashimoto’s thyroiditis (n = 3), and the duration of the disease ranged from 2–24 months. The diagnosis of TSH-secreting adenomas was based on clinical findings of goiter, hyperthyroidism, increased serum FT4 and TSH levels, and a pituitary mass on computed tomography and/or magnetic resonance imaging scans. The diagnosis of TSH-secreting adenomas was confirmed by immunohistochemistry performed after partial surgical resection of the adenomas.

Study design

Nine patients with primary hypothyroidism were studied before and after 3–24 months (10.1 ± 2.5) of L-T4 replacement therapy. The two thyroidectomized patients with TSH-secreting pituitary adenomas were studied during high-dose L-T4 replacement, when serum FT4 levels were high in both patients, and after 2 wk of L-T4 withdrawal, when serum FT4 levels decreased to the normal or low range. L-T4 (Euthyrox, kindly provided by Merck S. A., Rio de Janeiro, Brasil) replacement doses in primary hypothyroid patients ranged from 1.3–2.0 µg/kg·d. Determinations of in vitro serum TSH bioactivity before and after L-T4 replacement or withdrawal in each patient were run in the same bioassay.

Immunoconcentration of serum TSH

To eliminate interference in the TSH bioassay caused by factors present in serum, TSH was extracted from serum using polystyrene tubes precoated with a monoclonal antibody directed against an {alpha}ß-epitope of the TSH molecule (kindly provided by Dr. P. B. Romelli, Technogenetics, Milan, Italy), as previously described (14). The absolute amount of serum to be immunopurified varied from 0.75–37.5 ml according to TSH levels determined by the immunoassay. Several serum aliquots (0.75 ml) were incubated overnight in precoated tubes at 4 C and kept under slow shaking. After two wash steps with Tris-HCl buffer (pH 7.8), TSH was eluted from the tubes with guanidine hydrochloride 2 mol/liter (pH 3.2), immediately buffered with PBS 0.5 mol/liter (pH 9.0), dialyzed against hypotonic Hanks’ balanced salt solution (HBSS; without NaCl) (pH 7.5), and concentrated to a final volume of 0.5–1.5 ml by filtration (Centriprep centrifugal concentrators, cutoff 10 kDa; Millipore Corp., Bedford MA). The amount of immunoreactive TSH in the immunoconcentrate was measured by immunofluorimetric assay. Final mean recovery of TSH after these procedures was similar in controls and hypothyroid patients (60%) due to nonspecific losses, and not to selection of particular molecular isoforms of TSH, as already described (13, 14). Immunoconcentrated serum samples were kept at –70 C until they were diluted in hypotonic HBSS with BSA 0.4% (1:2 to 1:8) and bioassayed in triplicate.

TSH and thyroid hormone immunoassays

TSH was measured in duplicate by a sensitive third-generation immunofluorimetric assay developed in our laboratory (15). This assay uses an anti-ß-TSH monoclonal antibody with less than 1% reactivity for FSH, LH, and hCG, coupled to microtiter polyethylene plaques (antibody concentration 10 µg/ml). Standard TSH was the second international research preparation of human TSH (IRP-80/558, provided by NIBSC, Potters Bar, UK) diluted in TSH-free human serum prepared by affinity chromatography in a Sepharose column coupled to the anti-ß-TSH monoclonal antibody. A monoclonal antibody against the {alpha}-subunit of the glycoprotein hormones was labeled with europium and used as the second antibody, thus allowing the assay to detect only intact TSH. Fluorescence was measured in a time-resolved fluorometer (Delfia; Wallac Oy, Turku, Finland). The intraassay coefficient of variation was 4% (for TSH 2.5 mU/liter), the interassay coefficient of variation was 6%, and the sensitivity of the assay was 0.03 mU/liter. Normal reference values are 0.4–5.0 mU/liter. Serum FT4 and T3 (total) were measured by fluoroimmunoassay kits (Delfia), and normal reference ranges are 7.7–19.3 pmol/liter for FT4 and 1.1–3.1 nmol/liter for T3.

TSH bioassay

The biological activity of TSH was evaluated by measuring cAMP production in extracellular fluid of Chinese hamster ovary cells transfected with recombinant human TSH receptor (14, 16). The cells were harvested from Petri dishes using trypsin ethylene glycol bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid mixture and then seeded in 96-well plates (40,000 cells per well). Twenty-four hours after seeding, the cells were fed with fresh RPMI 1640 medium supplemented with glutamine (200 mmol), geneticin (400 µg/ml), and fetal calf serum (10%), and the assay was run 24 h later.

After the cells were washed with HBSS containing calcium and magnesium at room temperature, 90-µl TSH standard solutions or diluted samples of the immunoconcentrates with 10-µl isobutyl methylxanthine 0.5 mmol were incubated in a water bath at 37 C under slow shaking for 1 h. cAMP was measured in the medium collected at the end of incubation. TSH was measured by immunoassay in all dilutions used in the bioassay. The results of the biological assay were expressed as the biological to immunological ratios (B/Is) of immunopurified TSH samples, thus rendering an estimation of the biological potency of circulating TSH molecules (intrinsic TSH bioactivity) (13). The intraassay coefficient of variation was 13%, and the interassay coefficient of variation was 22%.

Concanavalin A (conA) lectin affinity chromatography

Lectins are proteins that bind only one or a few sugars with relative specificity, thus allowing inferences to be made about the presence of specific sugar residues or structures in complex oligosaccharides. Glycoproteins applied to the lectin conA are eluted in three general classes according to mannose content: 1) unbound glycopeptides that have bisecting, triantennary, and multiantennary complex structures, with low mannose content, corresponding to more mature TSH molecules; 2) weakly bound glycoproteins, that elute with 10 mmol {alpha}-methylglucopyranoside, and have biantennary complex or truncated hybrid oligosaccharides; and 3) firmly bound glycopeptides that elute with 300-mmol {alpha}-methylmannopyranoside and have high mannose or hybrid oligosaccharides, corresponding to less mature TSH molecules (17, 18, 19).

conA affinity chromatography of serum TSH was performed in all patients before and after L-T4 replacement therapy, as previously described (19). Briefly, 1-ml conA Sepharose was put on 5-ml columns and equilibrated with buffer containing 10 mmol/liter Tris HCl, 150 mmol/liter NaCl, 1 mmol/liter MgCl2, 1 mmol/liter MnCl2, and 1 mmol/liter CaCl2 (pH 8.0). After equilibration, 0.5-ml serum was loaded onto the column and allowed to interact with the lectin for 1 h at room temperature, under slow shaking. These columns were then placed inside 15-ml plastic tubes and centrifuged with 1 ml of the column buffer. This procedure was repeated eight times to elute unbound TSH, followed by 10 times with {alpha}-methylglucopyranoside 10 mmol/liter added to the buffer to elute weakly bound TSH, and four times with {alpha}-methylmannopyranoside 300 mmol/liter added to the buffer, to elute firmly bound TSH. Fractions were pooled and dried using a Speed Vac (GMI, Inc., Ramsey, MN), reconstituted with 1-ml assay buffer, and their TSH content was measured by immunoassay.

Ricin lectin affinity chromatography with and without neuraminidase treatment

Specimens [25-µl immunopurified samples and 100 µl phosphate buffer (PB) (pH 6.6)] were incubated with and without neuraminidase (10 mU for 4 h at 37 C) obtained from Clostridium perfringens (type X; Sigma-Aldrich, St. Louis, MO). Prior experiments have already demonstrated that under these conditions, almost all of the sialic acid residues were cleaved from TSH.

Ricinus communis binds specifically to exposed galactose residues, and the presence of sialic acid attached to galactose prevents this binding. Cleavage of the sialic acid residues by neuraminidase exposes the galactose, and the degree of sialylation can be assessed by the increase in the binding of TSH to ricin after treatment with neuraminidase (18, 19).

Briefly, columns containing 1-ml R. communis insolubilized on beaded agarose (RCA 120; Sigma Chemical Co., St. Louis, MO) were equilibrated with PB (pH 7.4) and 0.05% BSA. Specimens [25-µl immunopurified samples and PB 100 µl (pH 6.6)] with or without neuraminidase treatment were loaded onto the column with 0.5-ml PB-BSA 0.05% and allowed to interact for 1 h at room temperature, under slow shaking. Unbound TSH was collected by repeated centrifugation (nine times with 1-ml PB-BSA 0.05%). Bound fractions were eluted using the same procedure with PB-BSA 0.05% containing 200-mmol/liter galactose (Sigma-Aldrich). Unbound and bound fractions were pooled and dried using a Speed Vac; dried samples were solubilized in 1-ml PB-BSA 0.05%, and TSH was measured by immunoassay. The difference between the percentages of TSH bound to ricin without and with neuraminidase treatment represents the amount of sialylated molecules.

Statistical analyses

Statistical analyses were performed by Student’s paired and unpaired t tests, Wilcoxon signed rank test, and Fisher exact test, as appropriate. Correlations were calculated by linear regression analysis (Pearson or Spearman correlation). Statistical significance was set at P < 0.05. Results are expressed as mean or mean ± SE unless otherwise stated.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
In vitro serum TSH bioactivity in primary hypothyroidism before and after L-T4 replacement

All nine patients with primary hypothyroidism had markedly high-serum TSH levels (136.2 ± 40.6 mU/liter), low-serum FT4 levels (2.7 ± 0.8 pmol/liter), and low-serum T3 levels (0.5 ± 0.2 nmol/liter) before treatment. As expected, after L-T4 replacement therapy, serum TSH levels decreased (4.5 ± 0.7 mU/liter; P < 0.01, Wilcoxon), and both serum FT4 (14.4 ± 1.0 pmol/liter; P < 0.0001) and T3 levels (1.7 ± 0.1 nmol/liter; P < 0.0001, paired t test) increased significantly. Individual TSH levels after replacement therapy were in the normal range (0.5–4.0 mU/liter) in five patients (range 1.1–3.0) and slightly above in four patients (range 5.3–7.7).

The biological activity of TSH, expressed as the TSH B/I, was significantly decreased in hypothyroid patients compared with age and sex comparable normal controls (0.48 ± 0.1 vs. 1.1 ± 0.2, respectively; P = 0.004, t test) and increased significantly after L-T4 replacement (0.48 ± 0.1 vs. 0.80 ± 0.1; P=0.01, Wilcoxon). The values of TSH B/I after L-T4 replacement in patients with primary hypothyroidism were not different from controls (P = 0.14, t test). These data are shown in Fig. 1Go.


Figure 1
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  		FIG. 1. In vitro serum TSH bioactivity before and after L-T4 replacement therapy in patients with primary hypothyroidism. NS, not significant.

 
As shown in Fig. 2Go, a strong positive correlation was found between the absolute increment of in vitro serum TSH bioactivity ({Delta}B/I) and the absolute increment of serum T3 ({Delta}T3) after L-T4 replacement in patients with primary hypothyroidism (r = 0.81; P = 0.004, Spearman). No significant correlation was found between the absolute increments of in vitro serum TSH bioactivity ({Delta}B/I) and serum FT4 (r = –0.39; P = 0.30, Spearman).


Figure 2
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  		FIG. 2. Correlation between the absolute increments of in vitro serum TSH bioactivity ({Delta}B/I) and serum T3 concentrations ({Delta}T3) after L-T4 replacement in patients with primary hypothyroidism.

 
In the control group, there was a strong inverse correlation between in vitro serum TSH bioactivity and age (r = –0.71; P = 0.01, Pearson).

Effect of L-T4 replacement on TSH glycosylation

Mannose content of serum TSH. Determination of the mannose content of serum TSH through conA affinity chromatography showed a similar prevalence of unbound, weakly, and firmly bound TSH isoforms in hypothyroid patients before (23 ± 2.3%, 34 ± 2.3%, and 43 ± 3.8%, respectively) and after L-T4 replacement (19 ± 2.8%, 34 ± 2.8%, and 47 ± 1.9%, respectively) (0.38 < P < 0.99, paired t test) (Fig. 3Go). No correlation was found between mannose content of serum TSH and TSH bioactivity (r = 0.29; P = 0.24, Spearman).


Figure 3
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  		FIG. 3. Relative distribution of serum TSH according to mannose content in patients with primary hypothyroidism before and after L-T4 replacement therapy. Bars represent mean ± SE values. NS, not significant.

 
Sialylation of serum TSH. The degree of sialylation of serum TSH (i.e. the difference in the percentage of hormone binding to ricin without neuraminidase and after neuraminidase treatment), determined in eight of the nine patients studied, was elevated in hypothyroid patients compared with controls (47.0 ± 2.4% vs. 29 ± 4.3%; P = 0.002, t test). As shown in Fig. 4Go, TSH sialylation decreased significantly after L-T4 replacement therapy (47.0 ± 2.4% vs. 33.0 ± 4.3%; P = 0.02, Wilcoxon). No significant difference was found in the degree of sialylation between hypothyroid patients after L-T4 replacement and control subjects (33.0% vs. 29.0%; P = 0.50, t test).


Figure 4
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  		FIG. 4. Relative distribution of serum TSH according to sialylation degree in patients with primary hypothyroidism before and after L-T4 replacement therapy. Bars represent mean ± SE values.

 
A strong inverse correlation was found between the biological activity and degree of sialylation of serum TSH when all data (n = 16) from hypothyroid patients, both before and after L-T4 replacement, were analyzed (r = –0.85; P < 0.001, Pearson), as well as between the variations of biological activity ({Delta}B/I) and degree of sialylation of TSH ({Delta}sialylated TSH) after L-T4 replacement (r = –0.75; P = 0.03, Pearson).

The degree of sialylation of TSH tended to correlate inversely with serum T3 levels (r = –0.45; P = 0.08, Spearman), and TSH sialylation values below the mean were associated with serum T3 values above the mean (P = 0.06, Fisher), but no significant correlations were observed between the variations in the degree of TSH sialylation and serum thyroid hormone levels after L-T4 replacement.

Effect of decreasing serum thyroid hormone levels on in vitro serum TSH bioactivity in patients with TSH-secreting adenomas

In two patients with TSH-secreting pituitary adenomas who were on supraphysiological thyroid hormone replacement due to previous surgical or radioiodine thyroid ablation, serum FT4, T3, and TSH levels were elevated, and the biological activities of TSH in these patients were in the normal range. After 2 wk of L-T4 withdrawal, serum FT4 and T3 levels decreased from 23.2–6.4 pmol/liter and from 3.6–1.6 nmol/liter, respectively, and serum TSH levels increased from 75–90 mU/liter in patient 1. In patient 2, serum FT4 and T3 levels decreased and from 32.2–25.7 pmol/liter and from 3.9–3.7 nmol/liter, respectively, and serum TSH levels increased from 23–25 mU/liter. The decrease in serum FT4 and T3 levels was accompanied by a decrease in the TSH B/I values in both patients: TSH B/I decreased from 0.5–0.3 in patient 1 and from 1.8–1.0 in patient 2.


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
Previous reports of in vitro TSH bioactivity in patients with primary hypothyroidism have shown conflicting results (11, 12, 13), so we sought that this issue should be revisited with improved study design and methodology. In vitro TSH bioactivity was evaluated before and after L-T4 replacement in the same group of patients using a specific and sensitive TSH bioassay based on the generation of cAMP by Chinese hamster ovary cells transfected with the human recombinant TSH receptor. This assay has shown better sensitivity, specificity, and reproducibility than its immediate precursor, the FRTL-5 bioassay (13). In addition, our study is unique in the use of sex and age comparable controls. The strong inverse correlation between in vitro serum TSH bioactivity and age observed in normal subjects in our control group underscores the importance of controlling for age in studies of in vitro TSH bioactivity in humans, although this correlation should be confirmed in a larger sample of normal subjects. An age-related decline in the biological activity of a pituitary glycoprotein hormone has been recently described for serum LH (20).

The study of TSH bioactivity in patients with changing thyroid hormone levels should also allow an assessment of the relative roles of hypothalamic TRH and serum thyroid hormones as regulators of serum TSH bioactivity. In primary hypothyroid patients, TSH secretion is increased under the influence of low concentrations of serum thyroid hormones and increased hypothalamic TRH stimulation. In this "low thyroid hormone/high TRH" state, we found decreased in vitro TSH bioactivity. During L-T4 replacement, when both TRH and TSH secretions decrease as serum thyroid hormone concentrations increase, in vitro TSH bioactivity also increased. When the high-serum concentrations of thyroid hormones in two patients with TSH-secreting adenomas receiving supraphysiological doses of L-T4 (a "high thyroid hormone/low TRH" state) were lowered by short-term L-T4 withdrawal (a "decreasing thyroid hormone/increasing TRH" condition), in vitro serum TSH bioactivity also decreased. In these two patients, the decrease in in vitro serum TSH bioactivity was predominantly due to changes in TSH secreted by the tumor, and not by normal thyrotrophs in response to decreasing serum thyroid hormone concentrations, because circulating TSH levels during L-T4 withdrawal increased by only 20% in case No. 1 and did not change in case No. 2. The bioactivity of circulating TSH among patients with TSH-secreting pituitary adenomas is highly variable, from low to high values (21).

Altogether, our observations indicate that thyroid hormone is a positive regulator of serum TSH bioactivity in humans. In accordance, we found a strong correlation between the increments of in vitro serum TSH bioactivity during L-T4 replacement and the increments of serum T3 levels. On the other hand, changes in in vitro serum TSH bioactivity occurred in an opposite direction to the presumed changes in hypothalamic TRH secretion. Using the same TSH bioassay used in our study, a low in vitro serum TSH bioactivity that increased after L-T4 replacement, but not to the levels found in an undefined control group, has also been reported in patients with primary hypothyroidism (22).

The evidence for TRH as a major regulator of TSH bioactivity in humans derives mostly from an early study showing that serum TSH in patients with hypothalamic hypothyroidism has low biological activity, and that chronic TRH administration was able to increase TSH bioactivity and serum thyroid hormone concentrations (6). However, the concomitant increase in serum thyroid hormone levels, a potential confounding variable, was not considered in that study. In that same study, acute TRH administration to three patients with hypothalamic hypothyroidism modestly increased in vitro TSH bioactivity and serum T3 levels after 120–180 min, but in vitro TSH bioactivity failed to increase after 30–90 min of TRH administration in patients with hypothalamic hypothyroidism and normal subjects in two other studies (23, 24). In addition, we have recently shown that in vitro TSH bioactivity is also decreased in patients with pituitary hypothyroidism without hypothalamic involvement in Sheehan’s syndrome (a "low thyroid hormone/high TRH" condition), even when these data were now reanalyzed in comparison to a more rigorously comparable control group (data not shown).

In animals, no studies of serum TSH bioactivity have been reported so far. The only study of the bioactivity of in vitro secreted TSH in rats suggests that TRH and thyroid hormone deficiency could differentially regulate in vitro TSH bioactivity in these animals (25). Interestingly, hypothyroidism after thyroidectomy (a "low thyroid hormone/high TRH" condition) did not change the bioactivity of TSH secreted by pituitary incubates, but both "in vivo" pretreatment of normal and thyroidectomized animals with TRH for 24 h and "in vitro" addition of TRH increased the bioactivity of TSH secreted by pituitaries from normal and thyroidectomized animals into the incubation medium. The effects of adding thyroid hormones to the incubation medium on the bioactivity of secreted TSH were not evaluated in that study.

To investigate the molecular basis of decreased in vitro TSH bioactivity in primary hypothyroidism, we performed chromatography analysis of circulating TSH using two columns with different lectins, conA, and R. communis. These studies have shown that primary hypothyroidism did not change the prevalence of the three classes of TSH isoforms according to their mannose content, but it did increase the degree of TSH sialylation. A higher degree of TSH sialylation, which increases with the duration of the disease, has been shown in primary hypothyroidism, and increased sialylation of TSH has been shown to decrease its in vitro bioactivity (18, 21, 26). TSH secreted in vitro by mice pituitaries becomes more sialylated and less sulfated after prolonged hypothyroidism (27). Interestingly, both mannose content and degree of sialylation of serum TSH found in primary hypothyroidism are similar to those that we have recently reported in pituitary hypothyroidism due to Sheehan’s syndrome (10), indicating that TSH secreted under "high TRH and low thyroid hormone" conditions has decreased bioactivity due to increased sialylation.

In contrast, a higher mannose content of serum TSH has been shown in patients with hypothalamic hypothyroidism, but the degree of sialylation, measured in only three patients, was not different compared with controls (19). Clearly, studies with more patients and proper controls are necessary to define better the carbohydrate changes underlying decreased in vitro TSH bioactivity in patients with hypothalamic hypothyroidism. It is conceivable that the interplay between decreased hypothalamic TRH secretion, shifting the production of TSH isoforms from lower to higher mannose content TSH (more immature isoforms), and low serum T3 concentrations, presumably increasing terminal sialylation, could result in a different and more complex carbohydrate alteration compared with those found in "high TRH/low thyroid hormone" conditions, such as primary and pituitary hypothyroidism. It should be kept in mind that, as far as in vitro TSH bioactivity in serum represents the "sum" of individual biopotencies of the various circulating TSH isoforms, a same quantitative change in in vitro TSH bioactivity can result from different changes in the prevalence of these TSH isoforms.

The increment of in vitro TSH bioactivity during L-T4 replacement in hypothyroid patients correlated directly with the increments in serum T3 levels and in the T3:FT4 molar ratios, but not with serum T4 levels, and correlated inversely with the degree of TSH sialylation. These correlations support the idea that serum TSH bioactivity is positively regulated by serum T3 concentrations via decreasing intrapituitary TSH sialylation. The underlying cellular mechanisms responsible for the increase in sialic acid residues on TSH during hypothyroidism have been recently investigated by "in situ" hybridization studies showing that sialyltransferase mRNA increases in thyrotrophs of hypothyroid mice (28). This effect could result from a direct action of T3 in the upstream regulatory region of this sialyltransferase gene, which has several putative thyroid hormone responsive elements that could be regulated by changes in serum T3 levels (28, 29).


   Acknowledgments
 
We thank Professor Gilbert Vassart for providing and giving permission for us to work with Chinese hamster ovary cells transfected with recombinant human thyrotropin receptor (JP-26) cells; Dr. P. B. Romelli and G. Chiodoni (Technogenetics, Milan, Italy) for providing antibody-coated tubes; Dr. J. G. H. Vieira for providing the cyclic adenosine 5'-monophosphate assay; Drs. Mariana Rosa Borges and Silvia Correa for helping in the follow-up of the patients; Ms. Ivonete Alves Carvalhaes for blood collections; Ms. Samia Cavassani and Ms. Enza Giammona for technical assistance in the assays; and Dr. Luca Persani for technical discussions.


   Footnotes
 
This work was supported by Grant 98/10906-5 from Fundação de Amparo à Pesquisa do Estado de São Paulo, São Paulo, Brazil, and by a special fund from Centro de Estudos em Endocrinologia da Escola Paulista de Medicina. J.H.A.O. was the recipient of a scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior from 1998 to 2002.

This work was presented in part at the Seventh International Pituitary Congress, Phoenix, Arizona, June 23–25, 2001.

Disclosure Information: J.H.A.O. is now employed as a Medical Leader for Diabetes at Eli Lilly and Company. E.R.B., T.K., and J.A. have nothing to declare.

First Published Online May 15, 2007

Abbreviations: B/I, Biological to immunological ratio; conA, concanavalin A; FT4, free T4; HBSS, Hanks’ balanced salt solution; PB, phosphate buffer.

Received October 11, 2006.

Accepted May 7, 2007.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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