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Original Studies |
Institute of Endocrine Sciences, University of Milan, Istituto Auxologico Italiano Instituto di Ricovero e Cura a Carattere Scientifico (L.P.), and Ospedale Maggiore Instituto di Ricovero e Cura a Carattere Scientifico (S.B., G.F., P.B.-P.), 20145 Milan; and Department of Clinical Science, Endocrinology, University of Rome La Sapienza (E.F.), 00100 Rome, Italy
Address all correspondence and requests for reprints to: Luca Persani, M.D., Ph.D., Laboratorio di Ricerche Endocrinologiche, Istituto Auxologico Italiano Instituto di Ricovero e Cura a Carattere Scientifico, Via Ariosto 13, 20145 Milan, Italy. E-mail: persani{at}auxologico.it
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Abstract |
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4 mU/L, indicating
prevalent hCH, was found in the remaining 44%. Net TSH-I increments
showed significant correlation with basal FT4
(P < 0.02), indicating the relevance of pituitary
TSH reserve in the pathogenesis of CH. Circulating TSH was
immunoconcentrated and tested in bioassay and in ricin affinity
chromatography. The ratio between biological (B) and immunological (I)
activities of circulating TSH was reduced (n = 25; TSH B/I,
0.38 ± 0.19) compared to the values recorded in normal subjects
(n = 26; TSH B/I, 1.53 ± 0.54; P <
0.001) and primary hypothyroid patients (n = 24; TSH B/I,
0.74 ± 0.31; P < 0.001), but no difference
between pCH (n = 9; 0.36 ± 0.16) and hCH (n = 16;
0.39 ± 0.20) was seen. TSH B/I values in CH patients showed a
limited overlap with normal values (20%) and a highly significant
correlation with the FT3 response to endogenous
TRH-stimulated TSH (P < 0.005). The elevated
sialylation degree of TSH molecules may explain part of these
findings. In conclusion, the secretion of TSH molecules with reduced bioactivity is a common alteration in the patients with hypothalamic-pituitary lesions, contributing along with the impairment of pituitary TSH reserve to the pathogenesis of CH.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Twenty-six healthy volunteers and 24 patients with primary hypothyroidism (PH) served as control groups in TSH bioactivity and glycosylation studies. Blood was always drawn during the morning. The data for these subjects have been reported previously (18).
Patients with CH
Forty-one patients with different hypothalamic-pituitary
disorders (22 pituitary macroadenomas, 8 craniopharyngiomas or other
suprasellar tumors, 3 cranial irradiation, 1 hypophysitis, and 6
patients with a history of breech delivery and neonatal pituitary
insufficiency) were studied. All patients gave their informed consent
to the study. All patients had had a previous diagnosis of CH and were
negative for the presence of circulating antithyroglobulin and
antithyroperoxidase autoantibodies. On physical examination, thyroid
glands were normal or hypotrophic. All patients were studied during
appropriate replacement therapy for concomitant pituitary deficiencies,
with the exception of GH, and after a mean 60-day period of
L-T4 therapy discontinuation (19).
The period of L-T4 therapy
discontinuation was sufficient to induce clinical and biochemical
hypothyroidism [free T4
(FT4) levels were low in all patients; see
Results] and to restore levels of measurable TSH from the
undetectable TSH concentrations observed during substitutive
L-T4 therapy (19). The
patients were also studied during a TRH stimulation test (200 µg,
iv). The TRH test was performed to assess 1) the hypothalamic or
pituitary origin of CH [blunted TSH responses (<4 mU/L) were
considered indicative of a prevalent pituitary involvement;
normal/exaggerated TSH responses (4 mU/L) were considered indicative
of a prevalent hypothalamic lesion], and 2) the net
FT4 and FT3 increments
observed 2–3 h after TRH injection (n = 32). Blood for the
evaluation of circulating TSH bioactivity was drawn at baseline.
Immunoassays
Immunoreactive TSH (TSH-I) was measured by a third generation immunofluorometric assay (Delfia, Pharmacia, Milan, Italy), using a two-step procedure and TSH International Reference Preparation 80/558 as reference. The sensitivity of the TSH immunoassay is 0.007 mU/L, normal values range from 0.24–4.0 mU/L. Serum FT3 and FT4 levels were measured by direct back-titration methods, using Delfia technology (Pharmacia). Normal values for FT3 and FT4 immunoassays range between 4–8 pmol/L and 9–20 pmol/L, respectively. The interassay coefficient of variation is less than 5.0% in both cases. cAMP in incubation buffers was measured by commercial RIA (RIANEN, DuPont Co., Billerica, MA).
TSH bioassays
Immunoconcentration of circulating TSH. As human serum contains several factors that interfere in the TSH bioassay response, circulating TSH was immunopurified using polystyrene tubes coated with a monoclonal antibody directed against a conformational epitope, as previously described (20). The minimal amount of TSH (before immunoconcentration) necessary to perform a reliable estimation of its biological activity is 30 µU. The total volume of CH serum samples varied from 5–52 mL depending on the immunoreactive TSH concentrations. The recovery before the concentration step was 88–98%, whereas final recovery ranged between 52–68%; the discrepancy was due to nonspecific losses and not to selection of particular molecular forms of TSH (20). Furthermore, the possible presence in the purified samples of non-TSH substances able to alter cAMP production in the bioassays was ruled out by the observation of unmodified basal cAMP accumulation after incubation of the cells with the immunopurified material from a Graves’ patient with suppressed TSH secretion (18).
Chinese hamster ovary cells expressing human TSH receptor (CHO-R). The CHO-R strain JP-26 (supplied by Dr. G. Vassart, Brussels, Belgium) was used (21). Three different dilutions of immunoconcentrated TSH were bioassayed in triplicate, as was TSH standard. Extracellular cAMP was measured in the medium collected at the end of the incubation. The sensitivity of this system is 1.6 ± 0.25 mU/L. The other characteristics of the assay are similar to those previously reported (18, 20). The accumulation of cAMP was exclusively due to the stimulation of transfected TSH receptor in strain JP-26, as no stimulation was observed when immunopurified samples were tested in the control cells not expressing the TSH receptor (strain JP-02) (18).
As our experiments fulfilled the criteria illustrated by Chappel (22), i.e. the same reference preparation was always employed, and parallelism between immunoconcentrated sample dilutions and the TSH standard curve was observed in both immuno- and biological assays, the results of the TSH bioassay are expressed as the biological (B) to immunological (I) ratio (B/I) of immunoconcentrated TSH samples (mean ± SD; n = 9), thus giving an estimation of the biological potency of immunopurified materials.
Ricin lectin affinity chromatography
This investigation was performed as previously described (18, 23). Briefly, columns containing 1 mL Ricinus communis insolubilized on beaded agarose (RCA 120, Sigma, St. Louis, MO) 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 neuraminidase (NAM) treatment (10 mU, 4 h at 37 C) 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 under vacuum (Speed-Vac, Jouan, A.L.C. International, Cologno Monzese, Italy); dried samples were solubilized in 1 mL PB-BSA, and TSH was measured by immunoassay. The final recovery was always above 77%. 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 before and after NAM treatment represents an estimation of the amount of sialylated molecules (18, 23).
Statistical analysis
Data were analyzed by Student’s t test and ANOVA, as appropriate. Differences were considered statistically significant if P < 0.05. Results are expressed as the mean ± SD.
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Results |
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In the CH patients, immunoreactive TSH (TSH-I) ranged between
0.08–11.1 mU/L (mean ± SD, 2.6 ± 3.1; normal,
0.24–4.0), FT4 ranged between 0.6–8.8 pmol/L
(4.3 ± 2.6; normal, 9–18), and FT3 ranged
between 1.2–5.4 pmol/L (3.1 ± 1.1; normal, 4–8). Antithyroid
autoantibodies (antithyroperoxidase and antithyroglobulin) were absent
in all cases. No correlation between basal TSH and
FT4 values was found (Fig. 1
).
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An absent/impaired TSH response to TRH (net TSH-I increment, <4.0
mU/L), indicating a prevalent pCH, was found in 56% of the patients
(1.16 ± 1.02 mU/L; 0.06–3.84); in these cases serum
FT4 was 4.9 pmol/L or less. Net TSH-I increments
of 4.0 mU/L or more, indicating a prevalent hCH, were found in the
remaining 44% (10.3 ± 4.4 mU/L; 4.0–18.0). Serum
FT3 and FT4 increments in
response to TRH-stimulated endogenous TSH were absent/impaired (<1.1
pmol/L) in the large majority of CH patients (78%). Net TSH-I
increments after TRH injection showed significant correlations with
baseline TSH-I (r = 0.423; P < 0.02) and
FT4 (r = 0.376; P < 0.02)
concentrations (Fig. 2).
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Whenever the absolute amount of basal TSH-I was sufficient (n
= 25), we carried out the immunoextraction of TSH molecules to test the
immunopurified samples in the CHO-hTSHR bioassay. In the CH patients,
circulating TSH B/I was significantly reduced (range, 0.10–0.73; mean,
0.38 ± 0.19) compared to the values recorded in normal subjects
(n = 26; 1.53 ± 0.54; P < 0.001) and
primary hypothyroid patients (n = 24; 0.74 ± 0.31;
P < 0.001), but no difference between pCH (n = 9;
basal TSH-I: range, 0.8–2.3; median, 0.9 mU/L; net TSH-I increments
after TRH: range, 0.27–3.84; median, 1.30 mU/L) and hCH (n = 16;
basal TSH-I: range, 1.7–11.1; median, 3.30 mU/L; net TSH-I increments
after TRH: range, 4.0–18.02; median, 10.55 mU/L) was seen (TSH B/I:
pCH, 0.36 ± 0.16; hCH, 0.39 ± 0.20; P = NS;
Fig. 3). The TSH B/I values of CH
patients showed a limited overlap (20%) with the normal range and a
highly significant correlation with the net FT3
response to the stimulation of endogenous TSH after TRH injection
(r = 0.673; P < 0.005; Fig. 4).
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In five patients, the amount of immunopurified TSH was sufficient
to perform this analysis. They showed low ricin binding for untreated
TSH molecules, as in normal (n = 8) and PH (n = 10) controls,
whereas an increased amount of total TSH was bound to ricin after NAM
treatment, indicating an increased sialylation degree of carbohydrate
chains compared to normal or primary hypothyroid controls [CH, 63
± 12%; vs. normal subjects, 24 ± 6%
(P < 0.01); or vs. PH, 45 ± 8%
(P < 0.01); Table 1].
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In CH, the secretion of TSH with low bioactivity accounts for the lack of correlation between immunoreactive TSH and free thyroid hormone concentrations, and the absent/impaired increments in free thyroid hormone after acute stimulation of endogenous TSH by TRH (despite normal or even exaggerated TSH responses in patients with prevalent hypothalamic defects). In contrast, it is well known that bovine TSH administration was rapidly effective in stimulating thyroid hormone secretion and iodide uptake in patients with CH (1, 2, 9, 10, 11, 12, 13, 14, 15). The present findings are consistent with those obtained by means of different bioassays in a small number of selected patients with hypothalamic hypothyroidism (13, 14, 15, 24). Indeed, the relevant contribution of the low TSH bioactivity to determining CH in the majority of the patients with hypothalamic-pituitary disease is highlighted by the direct correlation between the circulating TSH B/I values and the net FT3 responses. It is conceivable that such a correlation could be even more significant if TSH B/I was measured 60–180 min after acute TRH injection. Interestingly, the recovery of a normal TSH B/I after acute TRH treatement was obtained in only one CH patient of six tested (15, 20). On the contrary, acute TRH administration to normal subjects was reported to release TSH isoforms with varied oligosaccharide structure (25) and lower bioactivity (26).
An animal model for hypothalamic hypothyroidism was recently generated by homologous recombination (16). As in our patients with hCH, the TRH knockout mice have blunted T3 responses to endogenous TRH-stimulated TSH despite exaggerated TSH increments and have elevated immunoreactive TSH levels in serum, a finding in contrast with the reduced number of thyrotropes in their pituitaries (16). The relevance of the impaired pituitary TSH reserve in the determination of CH in our patients is supported by the significant direct correlation between net immunoreactive TSH responses to TRH and basal FT4 concentrations; a similar finding was reported by Horimoto et al. (17), studying seven patients with CH of different origin. Interestingly, TRH treatment restored the normal number of thyrotrope cells in TRH knockout mice (16). In humans with hypothalamic hypothyroidism, daily administration of TRH for 1–4 months was transiently effective in restoring the secretion of bioactive TSH and euthyroidism (16, 27). A quantitative modification of the residual thyrotrope population might also occur in CH patients during chronic TRH.
Interestingly, the increased sialylation degree of TSH carbohydrate chains observed in CH patients is even higher than that previously found in primary hypothyroid patients (18) and may partially explain the diminished bioactivity of circulating molecules. Moreover, the terminal sialylation results in prolongation of the half-life of the hormone (9, 10, 11), which may justify the exaggerated and prolonged TSH responses to TRH and the maintenance of normal/high immunoreactive concentrations of TSH in hCH despite a reduction of producing cells. This finding is in agreement with data indicating that the transcription of some glycosyltransferases, such as sialyltransferase, is up-regulated in hypothyroidism (28). The increased sialylation degree may thus be a consequence of the hypothyroid state at the thyrotrope levels, but the mechanism accounting for the severe impairment of intrinsic TSH bioactivity in CH remains to be completely elucidated. It is likely that the synthesis and secretion rate of TSH are markedly increased in the remaining thyrotropes, resulting in a subversion of the posttranslational processing that is worsened by the concomitant impairment of TRH action (29, 30). In agreement, we and others (23, 31), have observed an increased amount of poorly processed TSH molecules with high mannose carbohydrate chains in the serum of some patients with hypothalamic hypothyroidism.
In conclusion, the secretion of TSH molecules with reduced bioactivity is a common alteration in patients with hypothalamic-pituitary lesions, contributing along with the impairment of pituitary TSH reserve to the pathogenesis of sporadic CH.
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Acknowledgments |
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Footnotes |
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Received December 27, 1999.
Revised March 17, 2000.
Accepted June 30, 2000.
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References |
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, single cases; •, mean ± SD)
and in control subjects (mean ± SD; 
, normal
subjects; 
, primary hypothyroid patients). The dotted
line indicates the lower limit of the normal range. Both groups
of CH patients have B/I ratios significantly lower than those observed
in normal or primary hypothyroid controls.