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Original Studies |
Neuroendocrine Unit, Division of Endocrinology, Hospital Sao Paulo-Universidade Federal de Sao Paulo (J.H.A.O., J.A.), Sao Paulo, Brazil 04039-002; and Institute of Endocrine Sciences, Ospedale Maggiore Istituto di Richerca e Cura a Caratere Scientifico and Istituto Auxologico Italiano, University of Milan (L.P., P.B.-P.), 20145 Milan, Italy
Address all correspondence and requests for reprints to: Julio Abucham, M.D., Division of Endocrinology, Hospital Sao Paulo-Universidade Federal de Sao Paulo, Rua Pedro de Toledo, 910 Sao Paulo, Brazil 04039-002.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Circulating TSH has multiple molecular forms or isoforms due to variations in the oligosaccharide structures (4, 5, 6, 7). TSH isoforms have been shown to possess different biological activities, and both increased and decreased TSH bioactivities have been reported in several thyroid disorders (8, 9). In central hypothyroidism due to various hypothalamic-pituitary conditions, serum immunoreactive TSH is usually normal or slightly increased, but possesses decreased biological activity (10, 11, 12, 13). Chronic TRH administration has been shown to increase TSH bioactivity and restore thyroid function in this condition (11). In primary hypothyroidism and in TSH-secreting pituitary adenoma patients, normal, reduced, and increased TSH bioactivities have all been reported (8, 12, 14, 15, 16, 17), whereas in thyroid hormone resistance patients, TSH bioactivity is increased (18).
We have recently shown that patients with hypothyroidism due to postpartum panhypopituitarism (Sheehan’s syndrome), a condition that follows massive necrosis of the anterior pituitary gland (19, 20), have unexpectedly normal or elevated TSH levels (21). In addition, TSH secretion was shown to be increased due to increased tonic, but not pulsatile, TSH secretion, and TSH circadian rhythm was severely blunted in Sheehan’s syndrome patients (21, 22). In this study we further investigated the paradox of hypothyroidism with increased TSH levels in Sheehan’s syndrome by determining the biological activity and the glycosylation pattern of serum TSH in this condition.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Nine women with Sheehan’s syndrome (age range, 34–61 yr; mean,
48.9 yr) and 11 healthy control subjects (8 women and 3 men; age range,
28–43 yr; mean, 33 yr) were studied. Informed consent was obtained
from all patients after approval of the study protocol by the Hospital
Sao Paulo-Universidade Federal de Sao Paulo ethical committee. The
diagnosis of Sheehan’s syndrome was based on clinical findings of
panhypopituitarism in patients with a positive history of massive
postpartum uterine bleeding followed by failure of lactation and
amenorrhea. The time elapsed between the last delivery and the
diagnosis ranged from 4–26 yr (median, 15 yr), and no patient had
received hormone replacement therapy before the study. Computerized
tomographic or magnetic resonance scans showed normal-sized empty
sellas, and dynamic testing of pituitary function showed blunted
responses of GH and cortisol to insulin-induced hypoglycemia (0.1 IU/kg
BW, iv), of PRL and TSH to TRH (200 µg, iv), and of LH to GnRH (100
µg, iv; Table 1
). Serum thyroid
autoantibodies were positive in a single patient who presented with
elevated antimicrosomal antibodies (1:6400). Control subjects were
healthy individuals with normal serum levels of TSH, thyroid hormones,
and thyroid autoantibodies. Control women were studied during the early
follicular phase of the menstrual cycle. Blood was collected in glass
tubes and allowed to clot at room temperature, and serum was separated
after centrifugation at 800 x g for 10 min. Serum
samples were kept at -20 C until assayed.
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To eliminate serum interference in the TSH bioassay, serum was
immunopurified and concentrated in polystyrene tubes precoated with a
monoclonal antibody directed against an 
ß epitope of the TSH
molecule (provided by Dr. P. B. Romelli, Technogenetics, Milan,
Italy) as previously described (12, 13, 16, 17, 18, 23). The
absolute amount of serum to be immunopurified varied from 7.5–37.5 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/L, pH 3.2, immediately buffered with 0.5 mol/L phosphate-buffered
saline, pH 8, dialyzed against hypotonic Hanks’ Balanced Salt Solution
(HBSS; without NaCl), and concentrated to a final volume of 0.5–1.5 mL
by filtration (Centriprep centrifugal concentrators; cut-off, 10
kDa; Millipore Corp., Bedford, MA). Immunoconcentrated
samples were kept at -20 C until bioassayed. The amount of
immunoreactive TSH in the immunoconcentrate was measured by
immunofluorometric assay. The final mean recovery of TSH after these
procedures was 46% due to nonspecific losses and not to selection of
particular molecular isoforms of TSH (12, 23).
Immunoconcentrated serum samples were kept at -20 C until they were
diluted in hypotonic HBSS with 0.4% BSA (1:2 to 1:8) and bioassayed in
triplicate.
TSH immunoassay
TSH was immunoassayed by a third generation immunofluorometric assay (Delfia, Wallac, Inc., Turku, Finland), using TSH International Reference Preparation 80/558 as reference. The detection limit was 0.03 mU/L. The intra- and interassay coefficients of variation were less than 5.0% and less than 7.0%, respectively. Normal reference values were 0.4–4.0 mU/L.
TSH bioassay
The biological activity of TSH was evaluated by measuring cAMP production in extracellular fluid of CHO-R cells-JP26 (Chinese hamster ovary cells transfected with recombinant human TSH receptor) (12, 24). The cells were harvested from petri dishes using trypsin ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid mixture and then seeded in 96-well plates (40,000 cells/well). Twenty-four hours after seeding, the cells were fed fresh RPMI 1640 medium supplemented with glutamine (200 mmol/L), geneticin (400 µg/mL), and FCS (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 isobutylmethylxanthine (0.5 mmol/L) were incubated in a water bath at 37 C under slow shaking for 1.5 h. cAMP was measured in the medium collected at the end of incubation by RIA (NEN Life Science Products, Boston, MA). 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/I) of immunopurified TSH samples, thus rendering an estimation of the biological potency of circulating TSH molecules (intrinsic TSH bioactivity) (25). The bioactive TSH concentration was calculated by multiplying the B/I ratio by the concentration of TSH in serum as determined by the immunometric assay.
Con A 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 Con A are eluted
in three general classes according to mannose: 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/L
-methylglucopyranoside and have biantennary complex or truncated
hybrid oligosaccharides; and 3) firmly bound glycopeptides that elute
with 300 mmol/L -methylmannopyranoside and have high mannose or
hybrid oligosaccharides, corresponding to less mature TSH molecules
(26, 27, 28).
Con A affinity chromatography of serum TSH was performed in five
patients and six controls as previously reported (26).
Briefly, 1 mL Con A-Sepharose was put on 5-mL columns and equilibrated
with buffer containing 10 mmol/L Tris-HCl, 150 mmol/L NaCl, 1 mmol/L
MgCl2, 1 mmol/L MnCl2, and
1 mmol/L 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 at
room temperature until 1000 [time] g was reached, when
centrifugation was stopped and 1 mL column buffer was added. This
procedure was repeated 8 times to elute unbound TSH, followed by 10
times with 10 mmol/L -methylglucopyranoside
added to the buffer to elute weakly bound TSH and 4 times with 300
mmol/L -methylmannopyranoside added to the buffer to elute firmly
bound TSH. Samples of these 3 fractions were lyophilized and
reconstituted with assay buffer, and their TSH content was measured by
the immunometric assay. The final recovery of TSH was 45–95% from the
initial amount of TSH loaded in the column.
Neuraminidase (NAM) treatment
Specimens (25 µL immunopurified samples and 100 µL phosphate buffer, pH 6.6), were incubated with or without NAM from Clostridium perfringens (type X, Sigma, St. Louis, MO; 10 mU for 4 h at 37 C). Prior experiments have demonstrated that under these conditions almost all of the sialic acid residues were cleaved from TSH.
Ricin lectin affinity chromatography
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 NAM exposes galactose, and the degree of sialylation can be assessed by the increase in binding of TSH to ricin after treatment with NAM (26, 28, 29).
This investigation was performed as previously described (26). Briefly, columns containing 1 mL R. communis insolubilized on beaded agarose (RCA 120, Sigma) were equilibrated with phosphate buffer (PB; pH 7.4) and 0.05% BSA. Specimens (25 µL immunopurified samples and 100 µL PB, pH 6.6) with or without NAM treatment were loaded onto the column and allowed to interact for 1 h at room temperature. Unbound TSH was collected by repeated centrifugation (20 times with 1 mL PB-BSA). Bound fractions were eluted using the same procedure with PB-BSA containing 200 mmol/L galactose (Sigma). Unbound and bound fractions were pooled and dried using a Speed-Vac (JOVAN, Milan, Italy); dried samples were solubilized in 1 mL PB-BSA, and TSH was measured by immunofluorometric assay. Ricin lectin specifically binds to exposed galactose or N-acetylglucosamine residues, whereas NAM treatment specifically removes terminal sialic acid, thus exposing the underlying galactose residue. The difference between the percentages of TSH bound to ricin without and with NAM treatment represents the amount of sialylated molecules.
Statistical analysis
Statistical analyses were performed using Student’s
t test or Mann-Whitney U test, as appropriate. Correlations
were calculated by linear regression analysis. Statistical significance
was set at P 
0.05. Results are expressed as the mean
or the mean ± SE unless otherwise
stated.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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) and were significantly
decreased compared with control values (3.7 ± 0.4 vs.
14.0 ± 0.9 pmol/L, respectively; P < 0.0001, by
Mann-Whitney U test). As shown in Fig. 1, basal serum TSH levels in Sheehan’s patients were significantly
increased compared with control values (3.8 ± 0.8 vs.
1.8 ± 0.2 mU/L, respectively; P = 0.01, by
t test), but the biological activity of TSH, expressed as
the TSH B/I ratio, was significantly decreased in Sheehan’s patients
compared with that in the same controls (0.6 ± 0.1 vs.
1.5 ± 0.2; P < 0.001, by t test).
When the bioactive TSH concentration in serum was calculated by
multiplying the concentration of immunoreactive TSH in serum by its
biological activity ratio (B/I), no significant differences were found
between patients with Sheehan’s syndrome and controls (2.1 ± 0.6
vs. 3.0 ± 0.4 mU/L; P = 0.25, by
t test).
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A significant correlation was found between bioactive TSH concentrations and FT4 levels in serum of patients with Sheehan’s syndrome (r = 0.66; P = 0.05), but not between serum immunoreactive TSH and FT4 levels (r = 0.21; P = 0.59) or between intrinsic TSH bioactivity and FT4 levels (r = 0.56; P = 0.12).
Con A affinity chromatography of TSH showed a similar distribution
(0.3 < P < 0.6, t test) among
unbound, weakly, and firmly bound TSH in Sheehan’s patients (16%,
38%, and 47%, respectively) and controls (15%, 34%, and 52%,
respectively; Table 2 and Fig. 2). The degree of TSH sialylation,
i.e. the difference in the percentage of hormone binding to
ricin without NAM and after NAM treatment, was higher in patients than
in controls (55% vs. 29%; P = 0.002; Fig. 3). Individual distribution patterns and
recoveries of TSH without NAM and after NAM treatment are shown in
Table 3.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Immunometric assays measure the total amount of serum TSH, and bioassays reflect the sum of the biopotencies of the various circulating TSH isoforms. When TSH-containing samples are quantified simultaneously by immunometric and biological assays, the ratio between bioactivity and immunoactivity serves as an index of the overall potency of circulating TSH molecules (25). Thus, variations in the B/I ratio result from changes in the amount of biological activity per unit of immunological activity. The bioassay used in this study, based on the generation of cAMP by Chinese hamster ovary cells transfected with the recombinant human TSH receptor, has been shown to possess better sensitivity, specificity, and reproducibility than its immediate precursor, the FRTL-5 bioassay (12).
Although TSH intrinsic bioactivity was decreased in Sheehan’s patients, immunoreactive serum TSH concentrations were higher than those in controls, so that the resultant bioactive serum TSH concentration (the product B/I x I) in Sheehan’s syndrome was not different from that in controls. The observation that serum FT4 levels correlated significantly with bioactive TSH concentrations, but not with immunoreactive TSH levels or intrinsic TSH bioactivity in Sheehan’s patients, reflects the relevant role of bioactive TSH concentrations in residual T4 secretion in Sheehan’s patients. Thus, the paradox of hypothyroidism with increased serum immunoreactive TSH levels in Sheehan’s syndrome cannot be solved simply by showing that serum TSH has decreased intrinsic bioactivity. Actually, another paradox is posed by the observation that the bioactive TSH concentration in serum, albeit normal, fails to sustain normal T4 levels in Sheehan’s patients.
To investigate the molecular basis of decreased TSH bioactivity in Sheehan’s syndrome, we performed chromatography analysis of circulating TSH employing two different lectins columns, Con A and R. communis. The distribution of circulating TSH isoforms according to mannose content in patients with Sheehan’s syndrome was similar to that in normal controls, but the degree of TSH sialylation was higher in Sheehan’s patients. Interestingly, the observed distribution pattern of mannose content of serum TSH in Sheehan’s syndrome is similar to that reported in patients with primary hypothyroidism and euthyroid controls, but different from that in patients with central hypothyroidism due to TRH deficiency, who showed an increased proportion of mannose-rich (less mature) TSH isoforms (26, 28). The reduced in vitro bioactivity and increased sialylation of TSH observed in Sheehan’s patients are in agreement with studies showing that increased sialylation of TSH decreases its in vitro bioactivity (7, 26, 28, 29, 30, 31).
Although increased serum TSH with low/undetectable intrinsic bioactivity is a common feature of primary, pituitary, and hypothalamic hypothyroidism, changes in the mannose content of serum TSH have only been observed in hypothalamic hypothyroidism (26, 28). In contrast, increased sialylation of serum TSH, as observed in our patients with pituitary hypothyroidism, has also been shown in primary hypothyroidism (29, 32), but not in the few patients with hypothalamic hypothyroidism studied to date (26, 28). Altogether, these observations indicate that TRH, but not T4, is necessary for the processing of high mannose (less mature) to low mannose (mature) TSH isoforms. On the other hand, when TRH secretion is increased and T4 levels are low, as in primary and pituitary hypothyroidism, sialylation of TSH increases and reduces its intrinsic bioactivity (29, 31). The messenger ribonucleic acid levels of sialyltransferase, an enzyme responsible for sialylation of exposed galactose residues of TSH, as well as messenger ribonucleic acid levels of galactosyltransferase and mannosidase II, which are enzymes that promote galactose incorporation and mannose processing, respectively, have all been shown to increase within thyrotrophs of propylthiouracil-induced hypothyroid mice (33, 34).
Our findings of increased serum levels of TSH with decreased intrinsic bioactivity resulting in normal bioactive TSH concentrations in patients with Sheehan’s syndrome may represent a late development after pituitary necrosis. The expected sequence of events affecting the hypothalamic-pituitary-thyroid axis after massive postpartum pituitary necrosis should start with a marked decrease in the serum concentrations of TSH followed by declining levels of T4, which, in turn, would stimulate TSH synthesis and secretion in the remaining thyrotrophs by acting both directly and indirectly at the hypothalamic paraventricular nuclei to promote increase TRH synthesis and secretion (35). In addition, the low levels of cortisol as well as a possible decrease in hypothalamic somatostatin due to decreased GH secretion could contribute to further increase TSH release in these patients. Under such circumstances, a higher proportion of sialylated TSH, which has reduced intrinsic bioactivity and decreased metabolic clearance rate, would be secreted (36) and further increase serum TSH levels. Increased serum TSH levels would eventually compensate for its low intrinsic bioactivity, increasing bioactive TSH concentrations toward the normal range, as we observed in our hypothyroid Sheehan’s patients long after pituitary necrosis occurred. Increasing bioactive TSH concentrations would increase the low T4 levels, but as serum T4 levels start to increase, thyrotrophs would, in turn, start to decrease TSH secretion, and bioactive TSH concentrations would decline. Considering the markedly reduced population of thyrotrophs in Sheehan’s syndrome, the expected TSH-lowering effect of similar increases in T4 levels should be much greater in hypothyroid Sheehan’s patients than in primary hypothyroidism patients. Indeed, normalization of serum FT4 levels to the midnormal range during T4 replacement therapy results in low/undetectable serum TSH levels in patients with central hypothyroidism (21), whereas physiological T4 replacement in primary hypothyroidism decreases TSH levels to the normal range. In addition, as those oscillations of circulating T4 and TSH in patients with Sheehan’s syndrome would occur within the subnormal range of T4 levels, where the dose-response curve of TSH inhibition by T4 is very steep (37), a vicious cycle would be generated, preventing normalization of serum T4 levels in these patients.
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Acknowledgments |
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Footnotes |
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Received January 14, 2000.
Revised October 6, 2000.
Revised December 6, 2000.
Accepted December 18, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunit:
evidence for secretion of TSH with increased bioactivity. J Clin
Endocrinol Metab. 62:704–711.-mannosidase-II messenger
ribonucleic acid levels increase with different kinetics in thyrotrophs
of hypothyroid mice. Endocrinology. 135:1980–1985.[Abstract]
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