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Dipartimento di Endocrinologia e Metabolismo, Ortopedia e Medicina del Lavoro (F.C., A.P., P.C., E.V., S.B., A.P., C.M.), Dipartimento di Oncologia (P.V., A.G.N.) e Dipartimento Chirurgia (P.M.), Università di Pisa, 56125 Pisa, Italy; and Endocrine-Hypertension Division, Department of Medicine, Brigham and Women’s Hospital (O.K., E.M.B.), Boston, Massachusetts, 00000
Address all correspondence and requests for reprints to: Claudio Marcocci, M.D., Dipartimento di Endocrinologia e Metabolismo, Ortopedia e Medicina del Lavoro, Università di Pisa, Via Paradisa 2, 56125 Pisa, Italy. E-mail: c.marcocci{at}endoc.med.unipi.it
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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It is well known that in patients with PHPT a greater concentration of Ca2+ is required to inhibit PTH secretion (2, 3, 4, 5, 6, 7, 8), but the molecular basis for this abnormality remains to be elucidated. The possibility that alterations in the expression and/or function of the CaR could be involved in this phenomenon has been the subject of active investigation (11, 12). No mutations in the coding sequence of the CaR gene have been identified in parathyroid tumors (10, 13), but a reduced messenger ribonucleic acid (mRNA) expression of a CaR promoter has been reported in a recent study (14). Moreover, a lower expression of CaR mRNA and CaR protein, compared with that in normal parathyroid tissue, has been reported in parathyroid adenomas and hyperplastic glands from patients with uremic hyperparathyroidism (15, 16, 17).
In the present study we evaluated the expression of CaR protein in a large series of sporadic parathyroid adenomas and investigated the relationship between the level of CaR expression and several clinical parameters, including the set-point of Ca2+-regulated PTH secretion measured in vivo.
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Subjects and Methods |
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The study group included 36 patients with sporadic PHPT, including 10 men, aged 33–70 yr (mean, 50 yr), and 26 women, aged 27–76 yr (mean, 55.7 yr). The diagnosis of sporadic PHPT was based on elevated Ca2+ (>1.32 mmol/L) and PTH (>65 pg/mL) levels and a negative family history.
Twenty-nine patients and 10 normal subjects (8 women and 2 men) participated in the PTH-Ca2+ set-point study. Patients were admitted after an overnight fast to the Department of Endocrinology between 0700–0900 h. Patients reclined for 30 min to stabilize levels of plasma proteins and calcium, and an iv catheter was placed in each arm, one for blood sampling and the other for calcium infusion. An electrocardiogram was performed before the infusion. After the equilibration period, 5 mL blood were drawn at 60 and 30 min before and immediately before initiating the calcium infusion. The mean Ca2+ and intact PTH concentrations at these time points were considered the basal value for subsequent data analysis. At time zero, calcium gluconate mixed in saline was infused iv for 3 h at a rate of 3 mg elemental calcium/kg·h, and blood samples were taken every 30 min for measurement of Ca2+ and intact PTH. The PTH-Ca2+ set-point was defined as the serum Ca2+ concentration corresponding to half-maximal inhibition of basal PTH. The relationship between serum Ca2+ changes and PTH secretion was analyzed using the four-parameter model, as previously described (7).
Serum ionized calcium was measured using a NOVA 8 calcium analyzer (Nova Biomedical, Waltham, MA). All values were adjusted to pH 7.40 (normal range, 1.13–1.32 mmol/L). Intact PTH was measured using an immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA; normal range, 10–65 pg/mL).
The decision to recommend parathyroidectomy was based upon the guidelines established by the Consensus Development Conference of the Management of Asymptomatic Primary Hyperparathyroidism (18).
The local ethical committee approved the study. Both tumor and blood samples were obtained after obtaining informed consent from all patients.
Parathyroid specimens
Pathological parathyroid specimens obtained at the time of
surgery were snap-frozen in liquid nitrogen after surgical excision and
were stored at -80 C until analysis. By assuming that the adenoma was
ellipsoid, the volume was calculated using the following formula:
(x x y x
z)x
/6, where x,
y, and z are the three-dimensional diameters in
centimeters. Biopsies of parathyroid glands from two normocalcemic
patients operated on for nodular goiter and normal bovine parathyroid
glands collected immediately after death were used as control tissues.
Histopathological examination by light microscopy was routinely
performed on each tissue sample using standard techniques.
Immunohistochemistry
A polyclonal CaR antibody, generated in rabbits with a peptide called FF7 (corresponding to amino acids 345–359 within the predicted extracellular domain of bovine parathyroid CaR) (10, 19) was used. Five-micron-thick sections of either normal or pathological glands were prepared in a cryostat and stored at 45 -80 C before use. In each experiment normal gland sections were included as controls, and staining of all normal and abnormal glands was performed simultaneously using the same reagents, the same batch of antiserum, and the identical incubation and washing times, as described below. Frozen sections were air-dried and then fixed for 8 min in acetone at -20 C, and a minimum of three to five sections were stained and analyzed for each parathyroid specimen, as described below. After treatment for 10 min at room temperature with an inhibitor of endogenous peroxidase (DAKO Corp., Carpinteria, CA), the sections were incubated with a blocking solution (1% BSA and 0.05 sodium azide) for 60 min at room temperature. The slides were then exposed to the CaR antibody for 17–18 h in blocking solution in a humidified chamber at 4 C (20, 21). They were rinsed three times with phosphate-buffered saline (PBS) and incubated with peroxidase-coupled, goat antirabbit secondary antibody for 1 h at room temperature. After washing three times with PBS, peroxidase staining was developed using the DAKO Corp. AEC Substrate System. The slides were subsequently mounted with coverslips and examined by light microscopy. For each sample consecutive sections mounted on the same slide were incubated with the original anti-CaR antibody or with the antibody preabsorbed with the peptide against which it was raised. All adenomas were examined in a single experiment, which was repeated at least three times. Normal human and bovine parathyroid sections, which were used as controls, were included in each experiment.
Each section was evaluated by two independent observers (F.C. and P.C.) at x400 magnification. The presence of a distinct staining along the cell surface of chief cells in a rim-like distribution, which was abolished by preabsorption of the CaR antibody with the specific peptide, was considered specific. The overall intensity of specific staining was scored as negative (-), weak (+), moderate (++), or strong (+++), and the adenomas were grouped in four categories, accordingly; in the case of +++ staining, the pattern and intensity of staining were similar to those of control tissues. Samples scored as negative showed irregular and faint staining, which was no different from when the section was incubated with the antibody preabsorbed with the specific peptide. In cases where the assessment of intensity of staining differed between the two observers, the disagreements were resolved by reaching a consensus after joint review using a conference microscope.
Western blot analysis
Tissue specimens were homogenized in ice-cold lysis buffer [50 mmol/L Tris-HCl (pH 7.4); 0.25 mol/L sucrose; 1 mmol/L ethyleneglycol-bis-(B-12-aminoethyl ether)-N,N,N',N'-tetraacetic acid and ethylenediamine tetraacetate; 10 µg/mL aprotinin, leupeptin, and calpain inhibitor; 40 µg/mL bestatin; and 100 µg/mL Pefabloc (Roche, Indianapolis, IN)]. Nuclei and cell debris were removed by low speed centrifugation (800 x g). The supernatant was then sedimented at 45,000 x g for 1 h, and the pellet was solubilized with 1% Triton X-100 in 100 µL lysis buffer and mixed with 2 x Laemmli sample buffer (22) containing 1 mmol/L dithiothreitol. Proteins (10 µg) were separated by SDS-PAGE (22) and transferred to nitrocellulose filters (Schleicher & Schuell, Inc., Keene, NH). After incubation for 1 h with blocking solution (PBS, 0.25% Triton X-100, and 5% dry milk), the filters were incubated in a humidified chamber at 4 C for 15–17 h with the same anti-CaR antibody as that used for immunohistochemistry and were subsequently developed using an enhanced chemiluminescence system (NEN Life Science Products, Boston, MA).
Statistical analysis
All data are expressed as the mean ± SD. ANOVA was used to analyze the statistical differences between several clinical and biochemical parameters in the four groups of adenomas, classified according to the intensity of CaR immunostaining, and significance was assessed by Fisher’s test. Simple linear regression was used to analyze the relationship between the PTH-Ca2+ set-point and basal Ca2+ and PTH levels and adenoma volume. An unpaired t test was used to evaluate the difference between PTH-Ca2+ set-point values of PHPT patients and controls. P < 0.05 was taken to indicate a statistically significant difference.
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Results |
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The relevant clinical and laboratory data are summarized in Table 1
. Each patient had a single parathyroid
adenoma. Preoperative concentrations of serum ionized
Ca2+ and intact PTH ranged between 1.32–1.87
mmol/L (mean, 1.49 ± 0.13 mmol/L) and 62–1490 pg/mL (mean,
202 ± 252 pg/mL), respectively. The volumes of the adenomas
ranged from 0.16–9.82 mL (mean, 1.18 ± 1.66 mL). A linear
correlation was found between adenoma volume and baseline PTH (r =
0.87; P = 0.0001) and Ca2+
(r = 0.4; P = 0.008) levels. In 35 cases tumor
removal was followed by persistent normalization of serum
Ca2+ and PTH concentrations. One patient had
recurrence of hyperparathyroidism 1 yr after surgery (no. 6).
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Twenty-nine patients underwent the calcium infusion test
preoperatively to evaluate the PTH-Ca2+
set-point. During infusion, the serum Ca2+
concentration increased progressively in all patients (mean increase
above baseline, 0.2 ± 0.06 mmol/L). The PTH concentration
(expressed as a percent decrease compared with the basal value) showed
no significant change (<10%) in 3 cases (no. 11, 28, and 33) despite
a marked increase in the serum Ca2+ concentration
(from 1.67, 1.53, and 1.69 mmol/L to 1.94, 1.72, and 1.95 mmol/L,
respectively). Interestingly, these 3 patients were among those with
the highest preoperative Ca2+ and PTH
concentrations. A variable degree of inhibition of PTH secretion,
ranging from 21–73% (mean, 54 ± 15%), was found in the
remaining 26 cases. In these latter cases a successful fit of the
corresponding Ca2+ and PTH values was achieved,
and the PTH-Ca2+ set-point could be calculated
using the 4-parameter model (7) (Table 1
). In 10 normal
subjects the mean PTH-Ca2+ set-point was
1.23 ± 0.04 mmol/L (range, 1.18–1.29 mmol/L). In PHPT patients
individual PTH-Ca2+ set-points ranged from
1.38–1.93 mmol/L (mean, 1.53 ± 0.14 mmol/L; P =
0.0001 vs. normal subjects) and were significantly
correlated with basal Ca2+ levels (r = 0.96;
P = 0.0001) and adenoma volume (r = 0.5;
P = 0.01). On the other hand, no correlation was
observed between the PTH-Ca2+ set-point and basal
PTH levels or between the percent maximal PTH inhibition and adenoma
volume and basal PTH or Ca2+ levels.
Immunohistochemistry
The normal parathyroid tissue showed an intense and specific
staining along the cell surface in a rim-like distribution (Fig. 1A) and was scored as +++. In all but 5
(14%) parathyroid adenomas, which were similar to normal parathyroid
tissue in the intensity of their CaR immunoreactivity and were scored,
therefore, as +++, the intensity of staining was decreased in a uniform
pattern throughout the gland: ++ in 10 (27%) cases, + in 16 (45%)
cases, and - in 5 (14%) cases (Fig. 1, B and C). The specificity
of the staining was confirmed in each case by abolition of the staining
after preabsorption of the anti-CaR antibody with the specific peptide
against which it was raised (Fig. 1D).
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). These two bands were absent
when peptide-preabsorbed antiserum was used for blotting (data not
shown). The fainter bands with lower molecular weights present in most
adenoma samples, but not in normal parathyroid, most likely represent
degradation products of the CaR and partially disappeared when
preabsorbed CaR antibody was used. As indicated in Fig. 2, there was a
good correlation between the intensity of CaR immunoreactive bands on
Western blotting and the immunohistochemical scoring of individual
samples.
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The relationship between CaR expression and several clinical and
biochemical parameters is summarized in Table 2. ANOVA showed a statistically
significant difference between the mean PTH-Ca2+
set-point values (in the 26 cases in whom it could be estimated) in the
4 groups of adenomas classified according to the intensity of
immunostaining (P = 0.025; F = 3.78). In
particular, the mean PTH-Ca2+ set-point was
significantly higher in the group of patients whose parathyroid adenoma
was classified as negative at immunohistochemistry than in those
classified as weak or moderate. In contrast, there was no difference in
the 4 groups in preoperative serum Ca2+, PTH
levels, or adenoma volume, even though the Ca2+
concentration and adenoma volume tended to be higher in patients with
absent or marked decreases in CaR expression compared with the other
groups. Interestingly, CaR expression was markedly reduced in the 3
patients with nonsuppressible PTH during calcium infusion (no. 11, 28,
and 33).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The recent cloning of the CaR (9, 10) has greatly clarified the mechanism by which parathyroid cells sense extracellular Ca2+, and alterations in the receptor’s structure and/or level of expression have been actively sought as possible explanations for the above-noted abnormalities in Ca2+regulated PTH release in patients with HPTH (11, 12, 13). To date, in sporadic parathyroid adenomas no mutations in the CaR gene have been reported (11, 13). A recent study has shown a reduced level of expression of the mRNA for a CaR promoter (14), but the question of whether a mutation in the noncoding region of the CaR gene is the basis for this abnormality remains to be determined. Reduced levels of expression of otherwise apparently normal CaR mRNA and protein have been reported in pathological parathyroid glands from patients with PHPT in several studies (15, 16, 17). As expected, in our series there was also a variable decrease in CaR protein expression as assessed by immunohistochemistry in parathyroid tumors compared with normal parathyroid glands from normocalcemic subjects. Western blot analysis of membrane proteins isolated from normal and pathological parathyroid glands using the same CaR antibody shows that most of the protein labeled had a molecular mass compatible with that of the glycosylated CaR protein (Mr = 130–150). The fainter bands with lower molecular masses present in most parathyroid adenomas, but not in normal parathyroid, have been observed in other studies (15, 24) and were only partially abolished when preabsorbed CaR antibody was used. They may represent partially degraded CaR or nonspecific staining. The intensity of staining was not correlated with the basal levels of Ca2+ or PTH levels or with adenoma volume. In our series five parathyroid adenomas were scored as negative for CaR expression. This finding is at variance with the results of other studies that also used immunohistochemistry (15, 16) and may be related to difference in the method used to quantify protein expression. It should be pointed out, however, that in three of these cases, PTH levels in vivo were essentially nonsuppressible during calcium infusion, which would be consistent with the markedly abnormal levels of CaR expression in these glands. In any case, the question of whether the reduced expression of the CaR protein represents a primary or a secondary phenomenon is still a matter for discussion.
It is well known that in patients with PHPT there is an increase in the set-point of Ca2+-regulated PTH secretion, and it has been suggested that a reduction in CaR protein might account for this abnormality. Indeed, in vitro studies have shown that there is a correlation between the progressive loss of suppression of PTH secretion and a reduction of CaR mRNA and protein expression in bovine parathyroid cells in culture (19). Moreover, a relationship between set-point and number of abnormal CaR alleles has been reported in familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism (5, 25).
To address this issue we evaluated in vivo PTH secretion during calcium infusion and compared the results with the CaR protein expression in the parathyroid adenoma surgically resected from the same patient. Most of our patients showed an increase in the PTH-Ca2+ set-point compared with what has been reported in normal subjects (26), and baseline serum Ca2+ was the strongest predictor of this change. Moreover, there was a statistically significant difference between the mean PTH-Ca2+ set-point in the four groups of patients ranked according to the intensity of CaR immunostaining of the parathyroid adenoma. In particular, the mean PTH-Ca2+ set-point was higher in the group of patients with negative CaR staining, suggesting that the apparent lack of expression of CaR is associated with a more severe abnormality in the control of Ca2+-regulated PTH release. Finally, a significant correlation was observed between the PTH-Ca2+ set-point and adenoma volume, indicating that parathyroid hyperplasia may also play a role in the abnormality of PTH secretion in PHPT. This pathogenic mechanism may be particularly relevant in the group of adenomas with CaR staining (+++) similar to that of normal parathyroid.
In summary, the results of the present study are consistent with previous data showing a substantial reduction of CaR expression in parathyroid adenomas from patients with sporadic PHPT. Our data also suggest that there is a relationship between the apparent CaR protein expression and PTH-Ca2+ set-point abnormality, suggesting that a reduced receptor content might have an important role in the pathogenesis of PHPT.
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Acknowledgments |
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Footnotes |
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Received February 28, 2000.
Revised August 10, 2000.
Accepted August 24, 2000.
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