1
TRANSIENT
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ASSOCIATED TO PRIMARY HYPERPARATHYROIDISM IN AN ELDERLY PATIENT:
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CASE REPORT AND LITERATURE REVIEW
HYPERCORTISOLISM
AND
SYMPTOMATIC
HYPERTHYROIDISM
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Chiara Sabbadin1*, Gabriella Donà2, Luciana Bordin2, Maurizio Iacobone3, Valentina Camozzi1,
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Caterina Mian1, Decio Armanini1
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*Corresponding author:
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Chiara Sabbadin [email protected]
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11
1
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Italy;
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2
14
3
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Gastroenterology, University of Padua, Padua, Italy
Department of Medicine– Endocrinology, University of Padua, Via Ospedale 105, 35128 Padua,
Department of Molecular Medicine-Biological Chemistry, University of Padua, Italy;
Minimally
Invasive
Endocrine
Surgery
Unit,
Department
of
Surgery,
Oncology
and
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E-mail: Gabriella Donà [email protected]; Luciana Bordin [email protected]; Maurizio
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Iacobone [email protected] ; Valentina Camozzi [email protected] ; Caterina
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Mian [email protected] ; Decio Armanini [email protected] .
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1
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Abstract
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Context: Primary hyperparathyroidism (PHPT) is often discovered routinely on blood test, at a
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relatively asymptomatic stage. However many studies suggest different systemic effects related to
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PHPT, which could be enhanced by an abnormal cortisol release due to chronic stress of
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hyperparathyroidism. Being PHPT frequently found in the 6th to 7th decade of life, a careful and
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multifaceted approach should be taken.
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Patient: We report the case of an 80-yr old, hypertensive and diabetic man, with symptomatic PHPT
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and an incidental pulmonary embolism. The patient was effectively treated with hydration, zoledronic
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acid, cinacalcet and high-dose unfractionated heparin. Because of his multiple comorbidities, further
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investigations revealed biochemical and morphological data consistent with Cushing’s syndrome.
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Screening for mutations of MEN genes was negative. Parathyroid surgery was successfully performed,
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but during post-operative period patient’s conditions suddenly worsened because of a transient
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thyrotoxicosis, probably induced by a previous exposure to iodine load and/or the thyroid surgical
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manipulation. A short-term treatment with beta-blockers was introduced for symptomatic relief. Four
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months after surgery, calcium-phosphate balance and thyroid function were normalized and also
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cortisol alterations resolved, suggesting a probably stress-induced hypercortisolism related not only to
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aging and hospitalization but also to PHPT.
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Conclusion: Chronic hyperparathyroidism has been linked with increased all-cause mortality. A
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functional chronic hypercortisolism could be established, enhancing PHPT related disorders. Only
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parathyroid surgery has been demonstrated to cure PHPT and complications related, showing similar
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outcome between older and younger patients. However, the management of post-operative period
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should be more careful in fragile patients. In particular, the early recognition and treatment of a
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transient thyrotoxicosis secondary to thyroid surgical manipulation or previous iodine load exposure
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could improve a successful recovery. Due to the increase in prevalence and the evidence of many
45
related complications even in asymptomatic PHPT, evidence and expert opinion-based guidelines for
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surgical treatment of PHPT should be developed especially for elderly patients.
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Keywords: Primary hyperparathyroidism, elderly, transient hyperthyroidism, iodinated-contrast
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induced thyrotoxicosis, hypercortisolism
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3
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Background
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Primary hyperparathyroidism (PHPT) is the third most common endocrinopathy seen today, frequently
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found in the 6th to 7th decade of life. PHPT is predominantly a sporadic disorder, caused by a single
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adenoma in approximately 85% of cases, by multiple gland hyperplasia in 15% and rarely by
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parathyroid carcinoma. Less than 10% of cases are inheritable, frequently associated with multiple
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gland hyperplasia. The most common presentation of PHPT is asymptomatic hypercalcemia, routinely
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found on blood test. However, many patients may suffer from minimal symptoms such as asthenia,
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constipation, polyuria, hypertension and neuro-psychiatric complications1. In some cases a renal colic
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is the first presentation of the disease. In older patients, concomitant morbidity and
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polypharmacotherapy may worsen symptoms and complications, and impact the management of
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PHPT2.
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In this paper we report a complex case of an elderly patient whit symptomatic PHPT associated with
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multiple comorbidities, in particular the finding of a functional hypercortisolism, resolved only after
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parathyroid surgery. The post-operative period was also characterized by a symptomatic transient
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thyrotoxicosis, probably induced by a previous exposure to iodine load and/or the thyroid surgical
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manipulation. The early recognition and treatment of this condition improved final outcome.
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Case Report
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An 80-year old man was admitted to a general hospital for polyuria, vomiting weight loss, worsening
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asthenia, myalgia and progressive cognitive impairment. He had a personal history of hypertension
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and type 2 diabetes, treated with losartan and metformin respectively. On physical examination he was
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sleepy, hypertensive (upright blood pressure 150/100 mmHg), tachycardic (100 beats for minute),
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without fever. He did complain of dyspnea, dry skin and mucosa and muscle weakness, but none
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neurological alterations. Biochemical assays revealed hypernatremia (149 mmol/L), severe
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hypercalcemia (4.08 mmol/L), hypophosfatemia (0.62 mmol/L), elevated levels of PTH (252 ng/L),
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reduced vitamin D (32 nmol/L) and slight renal failure (urea 8.7 mmol/L, creatinine 112 µmol/L).
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Blood count, liver and thyroid function were normal (Table 1). The electrocardiogram did not show
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remarkable signs of hypercalcemia. A cranial computed tomography (CT) scan excluded acute
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cerebrovascular events. A CT pulmonary angiogram detected partial thrombosis in three segmental
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branches of the right upper lobe pulmonary artery. Doppler ultrasound (US) revealed a deep vein
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thrombosis of the left posterior tibial vein. The patient was treated with isotonic saline hydration,
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furosemide, supplementation of vitamin D and an injection of zoledronate 4 mg, with a mild
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improvement of hypercalcemia and related symptoms. Daily high-dose unfractionated heparin was
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also administered. The patient was then transferred to our Endocrine Unit and treated with cinacalcet,
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with decrease of PTH, calcemia and calciuria values. Amlodipine and insulin were also added for
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worsening hypertension and diabetes. Neck US revealed a hypoechoic vascolarized nodule
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(14.7x10.5x9 mm) at the lower pole of the left thyroid lobe, consistent with enlarged parathyroid (Fig.
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1). A sesta-MIBI scintigraphy showed a homogeneous tracer uptake over the thyroid in the early
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images (Fig. 2A) and a remaining modest tracer uptake in the lower left thyroid lobe in the later
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images (Fig. 2B). Dual-energy x-ray absorptiometry revealed an osteopenia. Because of multiple co-
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morbidities, further investigations were performed to exclude other endocrine disorders: urinary
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metanephrine and normetanephrine values were normal, while plasma morning ACTH and daily
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urinary free cortisol were increased with impaired circadian cortisol rhythm, evaluated in two different
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measurements; serum cortisol after 1 mg overnight dexamethasone administration was not suppressed
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(Table 1). Direct abdomen CT was negative for adrenal diseases. Pituitary magnetic resonance
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imaging (MRI) evidenced a round mass, about 4 mm, without enhancement after gadolinium injection,
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in the left lateral portion of adenohypophysis, consistent with microadenoma. Screening for mutations
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of MEN genes was negative. The other pituitary hormones were normal. 8 mg overnight
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dexamethasone test (DST) showed cortisol suppression greater than 80% compared to baseline values.
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Because of the patient’s general conditions and a probably stress-induced hypercortisolism, no other
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investigations were done, giving priority to the surgical resolution of PHPT. Bilateral neck exploration
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was performed with removal of the upper right parathyroid and both the lower and the upper left
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parathyroid gland, with sharp decrease of intraoperative PTH (from 1296 to 39 ng/L). The histological
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diagnosis was consistent with multiglandular hyperplasia. Subsequent laboratory studies demonstrated
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a rapid decrease of serum calcium and PTH values, treated with oral calcium and calcitriol. However,
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four days after surgery the patient developed a sinusal tachycardia, mild heart failure and agitation
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alternating with stupor: there was no evidence of infection nor of volemic imbalance. Further
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investigation evidenced suppressed TSH, elevated free T4 and T3 values (Table 1), with undetectable
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anti-thyroid and TSH receptor antibodies. Thyroid palpation was not painful. The post-operatory
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99mTcO4 scintigraphy showed a reduced tracer uptake in total thyroid, especially in the lower left
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thyroid lobe, consistent with an inflammatory area (Fig. 3). Suspecting interference by the iodinated
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contrast used for pulmonary angio-CT about a month before, urinary iodine excretion were measured,
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resulting significant elevated (935 µg/L, normal range 100-200). Considering a possible dual
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pathogenesis of thyrotoxicosis, both destructive and iodine-induced, the patient was treated with
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atenolol for symptomatic relief. Two months after surgery, thyroid function and ioduria were normal
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and β-blocker was progressively stopped; 1 mg overnight DST still showed no adequate cortisol
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suppression. After other two months, calcium-phosphate balance was normal and serum PTH was near
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the lower limit of normal, therefore calcium and calcitriol supplementation were continued. Adrenal
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function was also revaluated: plasma ACTH and both salivary and urinary cortisol were all normal; 1
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mg overnight DST was repeated showing adequate cortisol suppression (Table 1); pituitary MRI
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confirmed the presence of a microadenoma, now consistent with a non-secretory incidentaloma, in
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careful biochemical and radiological follow-up.
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Discussion
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We report an elderly patient presenting symptomatic hypercalcemia with a certain derangement of the
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hemodynamic and neuropsychiatric systems. The detection of elevated PTH levels in the context of
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hypercalcemia is diagnostic of PHPT. The patient was immediately treated with rehydration and
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bisphosphonates, in order to restabilize an euvolaemic state and decrease calcium levels. The addition
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of cinacalcet is useful before elective surgery in elderly patients, as it is well tolerated and reduces not
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only calcium but also PTH levels3. Parathyroid surgery is the only definite cure for PHPT, but the
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risks and benefits of surgery need to be extensively considered in the elderly, given their more fragile
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state and comorbidities. In some cases where surgery is not indicated, cinacalcet can be the most
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effective treatment. A recent study did not find significant differences in surgical management and
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outcome between older and younger patients who underwent parathyroid surgery4. Preoperative
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imaging with ultrasonography and scintigraphy may help in the decision of surgery, even their
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sensitivity drops in detecting multiglandular disease, as found in our case report.
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Our finding of a transient thyrotoxicosis after parathyroid surgery is interesting and a dual
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pathogenesis could be involved: a destructive thyroiditis and/or a iodine-induced hyperthyroidism. The
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first condition is fairly unknown and underestimated since the symptoms could be mild and masked by
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other postoperative events5. Thyrotoxicosis seems to be related to an increased release of thyroid
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hormones induced by surgical manipulation; it could be influenced by other mechanisms, like pre-
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operative hypercalcemic setting, pre-existing goiter and a difficult parathyroidal glands identification
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during surgical exploration6. In a previous case report hyperthyroidism due to Graves’ disease
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developed after parathyroidectomy in a patient with underlying autoimmune thyroiditis, probably
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being reactivated after thyroidal autoantigen released during neck surgery7. An increased prevalence of
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PHPT in patients with thyroid disorders is also reported. Our patient had an euthyroid goiter, without
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abnormal MIBI-uptake in pre-operative investigations nor previous or underlying autoimmune
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thyroiditis. Retrospectively, the only apparent risk factors were the goiter, the previous pronounced
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hypercalcemic condition and the occasional finding of a multiple glands disease.
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The second possible cause of this transient thyrotoxicosis could be related to the previous iodinated
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contrast media exposure, leading to hypersecretion of thyroid hormones. This phenomenon, known as
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the Jod-Basedow effect, usually develops over 2 to 12 weeks typically in patients underlying thyroid
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disease, the elderly and people living in areas of iodine deficiency. Exposure to a large iodine load,
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such as occurs with iodinated contrast studies, can also cause acute destructive thyroiditis in people
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without pre-existing thyroid disease8. TcO4-scintigraphy could not discriminate the cause of
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hyperthyroidism, since pertechnetate is trapped by thyroid, but not organified and the resulting tracer
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uptake may be reduced. As happened in our case, the assessment of urinary iodine concentration may
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be helpful9. The significant elevated ioduria found in our patient about one month after the pulmonary
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angio-CT, could have been influenced by the older age and the slight renal failure secondary to the
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severe hypercalcemia. Some researchers have investigated the incidence and the role of prophylactic
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measures to reduce the risk of iodine-induced hyperthyroidism, without conclusive findings. In a large
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prospective study of 788 patients undergoing cardiac angiography, none of 27 with a suppressed TSH
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at baseline developed overt hyperthyroidism, while only 2 patients with no apparent risk factors
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became hyperthyroid10. In our case the concomitant neck surgery could have been a precipitating
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factor in the pathogenesis of hyperthyroidism.
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Both these conditions are usually self-limited and anti-thyroid drugs are not indicated. However, a
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short-term treatment with β-blockers could be required for symptomatic relief, especially in fragile
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patients.
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Since chronic hyperparathyroidism has been linked with increased all-cause mortality, in
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asymptomatic and elderly patients the optimal timing for parathyroidectomy is controversial11.
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Cardiovascular complications, linked to mineral homeostasis disruption are the leading cause of this
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increased mortality12. However, several studies have shown a direct effect of PTH on cardiomyocytes,
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endothelial and smooth muscle cells, leading to structural and functional cardiovascular alterations,
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often reversible after successful parathyroidectomy13. The complexity of PTH functions is further
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highlighted by data suggesting a bidirectional link between PTH and the renin-angiotensin-
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aldosterone-system, playing a synergic role in enhancing metabolic and cardiovascular complications,
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as we recently exposed in a case report14. Several studies have also evidenced an altered hypotalamic-
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pituitary-adrenal (HPA) axis in PHPT, correlated to psychiatric symptoms, hypertension and
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potentially contributing to cortical bone damage15. In vitro evidence supports a stimulatory effect of
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PTH on cortisol secretion16 and of calcemia on ACTH release17. In vivo data show a hypercortisolism,
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loss of circadian rhythm and lack of cortisol suppression after low-dose DST in PHPT18, which are not
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always recovered after surgical cure, as happened in our case.
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Alteration of cortisol expression and its circadian variability are also typical of aging, hospitalization,
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psychiatric and stress conditions; especially in elderly, HPA axis reactivity is also influenced by
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physiological changes of the expression of 11- β hydroxy steroid dehydrogenases-1 and the sensitivity
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of glucocorticoid receptor in the tissues19. False-positive results of the 1 mg DST are quite common
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and influenced by absorption, liver or renal alterations and the use of alcohol or drugs inducing
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CYP3A4. Being PHPT a long-standing disease frequently affecting old patients, the activation of
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adrenal function seems to recall a functional hypercortisolism due to chronic stress. A failure of
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limiting HPA axis response to stress could enhance the damaging effects of prolonged exposure to
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stress hormones20.
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In our case the data suggestive of a Cushing’s syndrome were consistent with the patient’s
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comorbidities (hypertension, diabetes, thromboembolism); because of his fragile condition and the
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confounding factors, we decided to postpone further confirmatory test and specific therapies. The
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finding of persisting hypercortisolism two months after discharge seems to exclude alterations only
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due to hospitalization, while the subsequent normalization of HPA axis after four months is likely to
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be secondary to the resolution not only of the stressful condition but also of PHPT.
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This complex picture suggests that parathyroid surgery may improve the prognosis, normalizing also
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HPA axis, which could contribute to PHPT related pathologies, such as bone metabolism, psychiatric,
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metabolic and cardiovascular disorders21.
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Conclusions
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There is ample evidence that PHPT is associated with HPA axis alterations, which could be involved
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in the increased metabolic, cardiovascular and neuropsychiatric complications. A wide variety of
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medical therapies are available, ameliorating complications related to hyperparathyroidism and
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allowing to even obviate the need for surgery. However, only parathyroid surgery has been shown to
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cure PHPT and significantly reduce the related disorders. Surgical management and outcome seem to
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be similar between older and younger patients. A transient thyrotoxicosis is a fairly underestimated
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condition, which could be secondary to previous iodine load exposure for radiological investigations
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but also related to thyroid manipulation during parathyroid surgery. The early recognition and
212
treatment of this complication may increase a successful recovery, especially in elderly patients.
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213
In conclusion, due to the increase in prevalence and the evidence of many related complications even
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in asymptomatic PHPT, evidence and expert opinion-based guidelines for surgical treatment of PHPT
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should be developed especially for elderly patients.
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Consent
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Written informed consent was obtained from the patient for publication of this Case report and any
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accompanying images. A copy of the written consent is available for review by the Editor of this
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journal.
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List of abbreviations used
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PHPT: primary hyperparathyroidism
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CT: computed tomography
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US: ultrasound
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MRI: magnetic resonance imaging
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DST: dexamethasone test
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HPA: hypotalamic-pituitary-adrenal
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Competing interests
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The authors declare that they have no competing interests.
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Authors’ contributions
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CS and DA treated the patient and drafted the article. GD and LB participated in the analysis and
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interpretation of data. MI operated the patient and together with VC and CM critically revised the
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manuscript. All authors read and approved the final manuscript.
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Acknowledgements
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We acknowledge Ms. Sophie Armanini for English revision.
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References
1. Pyram R, Mahajan G, Gliwa A. Primary hyperparathyroidism: skeletal and non-skeletal
effects, diagnosis and management. Maturitas 2011, 70:246-255
2. Conroy S, Moulias S, Wassif WS. Primary hyperparathyroidism in the older person. Age
Ageing 2003, 32:571-578
245
3. Jacobs L, Samson MM, Verhaar HJ, Koek HL. Therapeutic challenges in elderly patients
246
with symptomatic hypercalcaemia caused by primary hyperparathyroidism. Neth J Med
247
2012, 70:35-38
248
4. Jannasch O, Voigt C, Reschke K, Lippert H, Mroczkowski P. Comparison of outcome
249
between older and younger patients following surgery for primary hyperparathyroidism.
250
Pol Przegl Chir 2013, 85:598-604
251
252
5. Rudofsky G Jr, Grafe IA, Metzner C, Leowardi C, Fohr B. Transient post-operative
thyrotoxicosis after parathyroidectomy. Med Sci Monit 2009, 15:CS41-43
253
6. Lindblom P, Valdemarsson S, Westerdahl J, Tennval J, Bergenfelz A. Hyperthyroidism after
254
surgery for primary hyperparathyroidism. Langenbeck’s Arch Surg 1999, 384:568-575
255
7. Walfish PG, Caplan D, Rosen B. Postparathyroidectomy transient thyrotoxicosis. J Clin
256
257
258
Endocrinol Metab 1992, 75:224-227
8. Calvi L, Daniels GH. Acute thyrotoxicosis secondary to destructive thyroiditis associated
with cardiac catheterization contrast dye. Thyroid 2011, 21:443-449
259
9. Bahn RS, Burch HB, Cooper DS, Garber JR, Greenlee MC, Klein I, Laurberg P, McDougall
260
IR, Montori VM, Rivkees SA, Ross DS, Sosa JA, Stan MN. Hyperthyroidism and other
261
causes of thyrotoxicosis: management guidelines of the American Thyroid Association
262
and American Association of Clinical Endocrinologists. Endocr Pract 2011, 17:456-520.
263
Erratum in: Endocr Pract. 2013;19:384
264
10.Hintze G, Blombach O, Fink H, Burkhardt U, Kobberling J. Risk of iodine-induced
265
thyrotoxicosis after coronary angiography: an investigation in 788 unselected subjects.
266
Eur J Endocrinol 1999, 140:264-267
11
267
11.Yu N, Donnan PT, Leese GP. A record linkage study outcomes in patients with mild
268
primary hyperparathyroidism: The Parathyroid Epidemiology and Aduit Research
269
Study (PEARS). Clin Endocrinol (Oxf) 2011, 75:169-176
270
12.Pilz S, Tomaschitz A, Drechsler C, Ritz E, Boehm BO, Grammer TB, Marz W. Parathyroid
271
hormone level is associated with mortality and cardiovascular events in patients
272
undergoing coronary angiography. Eur Heart J 2010, 31:1591–1598.
273
13.Osto E, Fallo F, Pelizzo MR, Maddalozzo A, Sorgato N, Corbetti F, Montisci R, Famoso G,
274
Bellu R, Lüscher TF, Iliceto S, Tona F. Coronary microvascular dysfunction induced by
275
primary hyperparathyroidism is restored after parathyroidectomy. Circulation 2012,
276
126:1031-1039
277
14. Sabbadin C, Cavedon E, Zanon M, Iacobone M, Armanini D. Resolution of hypertension
278
and secondary aldosteronism after surgical treatment of primary hyperaldosteronism. J
279
Endocrinol Invest 2013, 36:665-666
280
15. Gianotti L, Tassone F, Pia A, Bovio S, Reimondo G, Visconti G, Terzolo M, Borretta G. May
281
an altered hypotalamo-pituitary-adrenal axis contribute to cortical bone damage in
282
primary hyperparathyroidism? Calcif Tissue Int 2009, 84: 425-429
283
16. Mazzocchi G, Aragona F, Malendowicz LK, Nussdorfer GG. PTH and PTH-related peptide
284
enhance steroid secretion from human adrenocortical cells. Am J Physiol Endocrinol
285
Metab 2001, 280: E209-E213
286
17.Fuleihan GE-H, Brown EM, Gleason R, Scott J, Adler GK. Calcium modulation of
287
adrenocorticotropin levels in women – a clinical research study. J Clin Endocrinol Metab
288
1996, 81:932-936
289
18.Rajput R, Bhansali A, Bhadada SK, Behera A, Mittal BR, Sialy R, Khandelwal N. A pilot
290
study on hypothalamo-pituitary-adrenocortical axis in primary hyperparathyroidism.
291
Indian J Med Res 2009, 130:418-422
292
293
19. Armanini D. Corticosteroid receptors in lymphocytes: a possible marker of brain
involution? J Steroid Biochem Mol Biol 1994, 49:429-434
12
294
295
296
297
20. Aguilera G. HPA axis responsiveness to stress: implications for healthy aging. Exp
Gerontol 2011, 46:90-95
21. Tirabassi G, Boscaro M, Arnaldi G. Harmful effects of functional hypercortisolism: a
working hypothesis. Endocrine 2014, 46:370-386
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Legends
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300
Table 1
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Principal biochemical parameters of the patient before and after surgery for primary
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hyperparathyroidism.
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Figure 1
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Neck ultrasound evidences a hypoechoic mass (14.7x10.5x9 mm), vascularized by a branch of the
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inferior thyroid artery, at the lower pole of the left thyroid lobe, consistent with enlarged parathyroid.
307
308
Figure 2
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Parathyroid scintigraphy after sesta-MIBI administration: the early image shows a homogeneous tracer
310
uptake over the thyroid (A), the later image evidence a remaining modest activity in the lower left
311
thyroid lobe (B).
312
313
Figure 3
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99mTcO4 scintigraphy, performed 4 days after parathyroid surgery, evidences a reduced uptake in
315
total thyroid tissue, especially in the lower left lobe.
14
Table 1 Principal biochemical parameters of the
patient before and after surgery for primary
hyperparathyroidism
Parameter
Ca (mmol/L)
P (mmol/L)
PTH (ng/L)
ACTH (ng/L)
morning salivary cortisol (ng/mL)
late-night salivary cortisol (ng/mL)
daily urinary cortisol (nmol/24h)
serum cortisol after 1 mg DST
(nmol/L)
TSH (mIU/L)
FT4 (pmol/L)
FT3 (pmol/L)
DST: dexamethasone soppression test
Figure 1
Normal
range
2,1-2,5
0,8-1,4
5-27
10-50
2,6-15,3
0,1-5,2
30-193
1 month
before surgery
4,2
0,6
252
128
13,1
15,8
557
4 days after
surgery
2,5
0,7
<4
126
281
4 months
after surgery
2,2
1,1
12
38
12,2
3,4
80
<50
0,2-4
9-22
3,9-6,8
223
1,04
19,9
3,9
0,03
26,9
10,4
30
1,17
18,7
4,1
Figure 2
Figure 3
Figure 4