DOI: 10.1111/j.1365-2362.2010.02385.x
ORIGINAL ARTICLE Evaluation of serum parathyroid hormone-related peptide in hyperthyroid patients Luca Giovanella*,†, Sergio Suriano*, Franco Keller†, Giorgio Borretta‡ and Luca Ceriani* * Nuclear Medicine and Thyroid Centre, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland, †Clinical Chemistry and Laboratory Medicine, Ente Ospedaliero Cantonale, Bellinzona, Switzerland, ‡Endocrinology and Metabolic Diseases, Azienda Ospedaliera ‘Santa Croce e Carle’, Cuneo, Italy
ABSTRACT Background Hypercalcemia occurs in 10–20% of patients with hyperthyroidism, but its pathogenesis is still unclear. Aim To evaluate changes in parathyroid hormone-related peptide (PTH-rP) concentration in hyperthyroid patients compared with healthy controls. Methods We studied PTH-rP, parathormone (PTH) and ionized calcium levels in 153 hyperthyroid patients, and 89 control subjects. These variables were revaluated after attainment of euthyroidism with the antithyroid drug carbimazole for 6 months in a subgroup of 47 patients. Results Pretreatment PTH-rP and ionized calcium level were significantly higher in hyperthyroid patients than in controls, whereas an opposite trend occurred for PTH. All parameters normalized after carbimazole therapy. Conclusion Untreated hyperthyroid patients exhibited a significant elevation in serum ionized calcium and PTH-rP and a significant reduction in serum PTH levels when compared with healthy controls. Our data favoured the hypothesis of a direct involvement of PTH-rP in the pathogenesis of hypercalcemia in hyperthyroid patients. Keywords Graves’ disease, hyperthyroidism, ionized calcium, parathormone, parathormone-related peptide. Eur J Clin Invest 2011; 41 (1): 93–97
Introduction Hyperthyroidism is associated with mild hypercalcemia in 10–20% of patients, which usually subsides after successful treatment of hyperthyroidism [1,2]. The thyrotoxic hypercalcemia may be due to the activation of osteoclastic bone resorption by the excess thyroid hormone, but its pathogenesis is not yet completely clarified [3,4]. Recently, we came across a case where hypercalcemia was associated with an increase in parathormone-related peptide (PTH-rP) levels in a patient with Graves’ disease (GD) [5]. Measurable PTH-rP levels are found in up to 100% of patients with humoral hypercalcemia of malignancy (HHM), in 75% of patients with breast carcinoma metastatic to bone and in some hypercalcemic patients with miscellaneous cancers [6]. Additionally, PTH-rP has been occasionally identified in benign endocrine neoplasms and also in hyperplastic and adenomatous tissues from the thyroid [7–9]. However, no data are available until now about patients with hyperthyroidism. Therefore, the present study was undertaken to evaluate the PTH-rP
concentration and its relationships with calcium metabolism in hyperthyroid patients.
Subjects and methods Subjects In the current study, we used a process of experimental design to estimate the optimal sample size required to provide adequate statistical power at our chosen level of significance. Power calculations were conducted using tools provided by Emphron Informatics (http://www.emphron.com) and suggested that 85 specimens would give us ‡ 80% power to ensure a statistical precision of at least 5% to detect a variation of biochemical variables in different groups [10]. One-hundred and fifty-three patients with primary hyperthyroidism were recruited from the Division of Nuclear Medicine and 89 healthy controls were recruited from healthy blood donors at Oncology
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Table 1 Clinical and biochemical characteristics of untreated hyperthyroid patients and controls Patients
Controls
Total no.
153
89
No. males/females
46/107
19/70
Age (year)
34 (14–67)
31 (18–60)
ns
25Æ7 (23Æ2–30Æ7)
26Æ2 (24–28Æ5)
ns
14Æ9 (6Æ4–30Æ7)
4Æ4 (2Æ7–4Æ8)
< 0Æ001
41Æ5 (22Æ8–87Æ9)
15Æ4 (9Æ6–18Æ5)
< 0Æ001
< 0Æ1 (< 0Æ01–0Æ09)
1Æ25 (0Æ4–3Æ6)
< 0Æ001
9Æ6 (1Æ8–137)
0Æ5 (£ 0Æ3–1Æ0)
£ 0Æ001
)2
BMI (kg m ) )1
Free T3 (pmol L ) )1
Free T4 (pmol L ) )1
TSH (mUI L ) )1
TRAb (U L )
P value
BMI, body mass index; TSH, thyrotropin; TRAb, TSH-receptor antibody.
Institute of Southern Switzerland (Switzerland) from June 1st, 2008 to December 31st, 2009. As displayed in the Table 1, patients and controls were matched for ethnic group, age, gender and body mass index (BMI). Patients were clinically and biochemically hyperthyroid at baseline [i.e. elevated free thyroxine (fT4) and free triiodothyronine (fT3) and suppressed thyrotropin (TSH)]. Primary hyperthyroidism was due to Graves’ disease (GD) in all cases [i.e. diagnosed on the basis of elevated TSH-receptor antibody (TRAb), hypoechoic thyroid pattern at ultrasound (US) and homogeneous increase of the 99m Tc-pertechnetate thyroid uptake]. All patients were treated with carbimazole (with or without propranolol) as further detailed below. Forty-seven patients volunteered for a second study and were revaluated after 6 months of treatment by carbimazole. No patient or control had other medical illness, and none of them was taking any medications other than those mentioned for the patients in the experimental protocol.
Experimental protocol Patients attended the Division of Nuclear Medicine after an overnight fast of 10–12 h. Weight, height, pulse, and blood pressure were measured. Fasting blood samples were collected for the measurement of TSH, fT3, fT4, TRAb, thyroperoxidase antibody (TPOAb), parathormone (PTH), PTH-rP and ionized calcium. Full blood count, liver and renal function were also assessed. All patients underwent thyroid US and 99mTc-pertechnetate thyroid scan as previously described [11]. In the second study, TSH, fT3, fT4, TRAb, PTH, PTH-rP and ionized calcium were revaluated after 6 months of thyrostatic treatment by carbimazole (Neo-mercazole; Nicholas AG, Basel, Switzerland).
Laboratory methods Ionized calcium and pH were immediately measured simultaneously at 37 C using semiautomatic ICA1TM analyzer
94
(Radiometer A/S, Copenhagen, Denmark) as previously described [12]. Haemoglobin, packed cell volume, white cell count, platelets and liver and renal functions were measured by routine laboratory techniques. Remaining blood samples were transferred on ice immediately and centrifuged at 700 g at 4 C for 15 min, and the supernatants were stored at )80 C until analysis. TSH, fT3, fT4, TPOAb and PTH were assayed using chemiluminometric assays on fully automated Immulite 2000 platform (DPC, Los Angeles, CA, USA). For all assays, withinrun and between-day imprecisions in a clinically representative concentration ranges were < 8% and < 10% respectively [13]. The TRAb and the PTH-rP measurements were performed by a 2nd generation radiometric assay (TRAk Human; BRAHMS GmbH, Berlin, Germany) and a two-site immunoradiometric assay (ACTIVE PTH-rP; Diagnostic Systems Laboratories, Webster, TX, USA) respectively. Within-run and between-day imprecisions were 8% and 11% (at 5 U L)1 TRAb concentration) and 6% and 11% (at 5 pmol L)1 PTH-rP concentration) respectively [14,15]. The lowest detectable level of PTH-rP distinguishable from zero PTH-rP standard with 95% confidence was 0Æ5 pmol L)1. This value was determined by processing a complete five point calibration curve, controls and 12 replicates of the zero standard in multiple assays. Undetectable PTH-rP levels were arbitrarily reported at 0Æ49 pmol L)1 for statistical analysis.
Ethics The study protocol was approved by the Local Ethics Committee, and subjects gave informed written consent.
Statistical analysis Data were expressed as median (range). Different groups were compared using the Mann–Whitney U test. Correlations between PTH-rP and other variables were determined using Spearman’s rank correlation coefficient (rho). A P level < 0Æ05 was considered statistically significant. Reporting of the study conforms to STROBE statement along with references to STROBE and the broader EQUATOR guidelines [16].
Results Characteristics of subjects and study progress The characteristics of patients and controls at entry of the first study are shown in the Table 1. In this study, patients with GD were treated by carbimazole. The median (range) initial carbimazole dose was 40 (10–60) mg day)1 and the maintenance carbimazole dose was 10 (5–20) mg day)1. Propranolol (Inderal; AstraZeneca AG, Zug, Switzerland) was used to control tachycardia, when needed, in the first 4–6 weeks of carbimazole treatment at doses of 80 (40–160) mg day)1.
ª 2010 The Authors. European Journal of Clinical Investigation ª 2010 Stichting European Society for Clinical Investigation Journal Foundation
SERUM PTH-rP IN HYPERTHYROID PATIENTS
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Table 2 Serum parathormone-related peptide (PTH-rP), parathormone (PTH) and ionized calcium distributions in untreated patients and controls PTH
Ca2+
Patients (n = 153)
0Æ8 (< 0Æ5–1Æ9)
3Æ2 (1–6Æ7)
1Æ27 (1Æ21–1Æ47)
Controls (n = 89)
0Æ5 (< 0Æ5–0Æ5)
4Æ6 (2Æ9–6Æ1)
1Æ19 (1Æ20–1Æ24)
P value
< 0Æ0001
< 0Æ001
< 0Æ01
1·4
Ca++ (mM )
PTH-rP
1·5 1·3 1·2 1·1 1 0·9 0·8 0·7 0·6
Serum PTH-rP, PTH and ionized calcium
GD
Controls
7 6
Screening for immunological interferences on PTH-rP measurement
5 4 3 2 1 0
2 1·8 1·6
PTH-rP (pM)
Inaccuracies caused by interferences from both exogenous and endogenous immunoglobulins/antibodies remained a problem for immunoassays; as heterophile antibodies, endogenous immunoglobulins may interfere in immunoassays leading to falsely high or low results [17]. To exclude interferences on PTH-rP measurement by TRAb and TPOAb, we repeated the basal PTH-rP measurement after incubating 500 lL of each serum sample in heterophilic aantibody-blocking tubes (HABT; Scantibodies, Santee, CA, USA) at room temperature for 1 h. For each sample, we calculated the differences between the original PTH-rP value and the measurement obtained after HABT treatment, as previously described [18]. No significant differences were found in PTH-rP values before and after HABT treatment, excluding significant interferences.
Controls
8
PTH (pM)
As displayed in the Table 2 and Fig. 1 untreated GD patients had significantly higher concentrations of PTH-rP and ionized calcium (P < 0Æ0001 and < 0Æ01) and lower concentrations of PTH (P < 0Æ001) than controls. The PTH-rP levels are positively related with ionized calcium levels. No relationship was found between PTH-rP and TSH, fT3, fT4, TRAb, TPOAb and PTH levels respectively (Table 3).
GD
1·4 1·2 1 0·8 0·6 0·4 0·2
Follow-up study After carbimazole administration trough 6 months, all patients normalized fT3 and fT4 as well as PTH-rP, PTH and ionized calcium serum levels (i.e. P £ 0Æ01 with respect to baseline values for all variables) (Table 4).
0
GD
Controls
Figure 1 Serum parathormone-related peptide (PTH-rP), parathormone (PTH) and ionized calcium distributions in untreated patients and controls (horizontal bar, median; vertical bar, distribution range).
Discussion The adverse effects of hyperthyroidism on the skeleton were known before the advent of satisfactory treatment for hyperthyroidism. One of the first reports of hyperthyroid bone disease was in 1891 when von Recklinghausen [19] described the ‘worm eaten’ appearance of the long bones of a young woman
who died from hyperthyroidism. With the introduction of antithyroid drugs and radioiodine in the 1940s, clinically apparent hyperthyroid bone disease became less common. However, bone density measurements during the last decade have
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Table 3 Multiple regression analysis: relationship between parathormone-related peptide (PTH-rP) and other variables Variables
Coefficient
SE
t
P value
PTH
)0Æ03822
0Æ03978
)0Æ961
0Æ3413
0Æ60702
0Æ27925
2Æ174
0Æ0346
Free T3
)0Æ00164
0Æ01151
)0Æ143
0Æ8872
Free T4
)0Æ00154
0Æ00978
)0Æ237
0Æ7560
TSH
)0Æ00157
0Æ01245
)0Æ176
0Æ8453
TRAb
)0Æ00598
0Æ00424
)1Æ412
0Æ1643
TPOAb
)0Æ00193
0Æ00877
)0Æ237
0Æ7544
Ionized calcium
PTH, parathormone; TSH, thyrotropin; TRAb, TSH-receptor antibody; TPOAb, thyroperoxidase antibody.
Table 4 Serum parathormone-related peptide (PTH-rP), parathormone (PTH) and ionized calcium distributions in a subset of 47 hyperthyroid patients before and after carbimazole treatment Patients (n = 47)
PTH-rP
PTH
Ca2+
Before treatment
0Æ8 (0Æ5–1Æ9)
3Æ4 (1Æ3–6Æ7)
1Æ26 (1Æ21–1Æ47)
After treatment
0Æ5 (< 0Æ5–0Æ7)
4Æ6 (2Æ9–5Æ9)
1Æ18 (1Æ19–1Æ25)
P value
< 0Æ01
< 0Æ01
< 0Æ01
demonstrated that bone loss is common in patients with overt hyperthyroidism and to a lesser extent in those with subclinical hyperthyroidism, whether caused by thyroid hyperfunction or excessive doses of thyroid hormone [20,21]. Additionally, subclinical hypercalcemia is documented in up to one-fourth of patients with hyperthyroidism. Excluding cases of coexisting primary hyperparathyroidism, hyperthyroid hypercalcemia is characterized by normal or suppressed PTH levels and usually subsides after successful treatment of hyperthyroidism [11,22,23]. The cause of bone disease and hypercalcemia in the hyperthyroid patients is not yet completely clarified. During the past decade, it was known that osteoblasts express nuclear receptors for T3; however, so far receptors have not been demonstrated in osteoclasts [24–26]. Accordingly, the T3 was found to be unable to stimulate resorption in monocultures of osteoclasts [27]. In co-cultures of osteoclasts and osteoblasts, however, the T3 stimulates osteoclastic resorption on cortical bone slides, and both the number and the area of the resorption pits were increased [28]. Thyroid hormone stimulates osteoclastic bone resorption, through increased expression of receptor activator of nuclear factor kappa B ligand (RANKL) in osteoblasts; additionally, T3
96
proved to directly stimulate osteoclasts differentiation by up-regulation of c-fos protein in osteoclast precursor cells [29,30]. However, the ligands for PTH/PTH-rP receptors are the main stimulators of bone resorption through interaction within osteoblasts [31]. Interestingly, the results we obtained here proved, for the first time to the best of our knowledge, that a significant increase in serum PTH-rP occurs in hyperthyroid patients and completely normalized during effective antithyroid treatment. Additionally, while calcium and PTH-rP levels are strongly related, any relationship was found between PTH-rP and thyroid hormones in our series. Taken globally, our data suggest that a [PTH-rP]-mediated pathway plays an independent role in the pathogenesis of hypercalcemia in hyperthyroid patients. Acknowledgement This study was supported by the Research Advisory Board – Ente Ospedaliero Cantonale, Bellinzona, Switzerland. Disclosures/Conflict of interest The authors declare that they have no competing interests. Author contributions LG conceived the study and drafted the manuscript; SS clinically managed the patients; FK carried out the immunoassays; GB drafted the manuscript; LC performed statistical analyses and drafted the manuscript. Address Nuclear Medicine and Thyroid Centre, Oncology Institute of Southern Switzerland, Bellinzona CH-6500, Switzerland (L. Giovanella, S. Suriano, L. Ceriani); Clinical Chemistry and Laboratory Medicine, Ente Ospedaliero Cantonale, Bellinzona CH-6500, Switzerland (L. Giovanella, F. Keller); Endocrinology and Metabolic Diseases, Azienda Ospedaliera ‘Santa Croce e Carle’, Cuneo I-12100, Italy (G. Borretta). Correspondence to: Luca Giovanella, Nuclear Medicine and Thyroid Centre, Oncology Institute of Southern Switzerland, Bellinzona CH-6500, Switzerland. Tel.: +41 91 8118672; fax: +41 91 8118250; e-mail:
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