PTH Is a Promising Auxiliary Index for the Clinical Diagnosis of Aldosterone-Producing Adenoma

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1 Original Article PTH Is a Promising Auxiliary Index for the Clinical Diagnosis of Aldosterone-Producing Adenoma Lin-Xi Zhang, 1,2, * Wei-Jun Gu, 2, * Yi-Jun Li, 2 Yang Wang, 3 Wen-Bo Wang, 2 An-Ping Wang, 2 Lei Shen, 2 Li Zang, 2 Guo-Qing Yang, 2 Zhao-Hui Lu, 2 Jing-Tao Dou, 2 and Yi-Ming Mu 2 BACKGROUND Parathyroid hormone (PTH) stimulates aldosterone secretion in human adrenocortex and is regulated by the renin-angiotensinaldosterone system. We speculated that measurement of PTH may be a valuable aid in the diagnosis of aldosterone-producing adenoma (APA). METHODS To test this hypothesis, we recruited 142 patients with adrenal adenoma, of whom 84 had an APA and 58 had a nonfunctioning adrenal adenoma (NFA). Plasma levels of intact PTH, serum potassium, sodium, calcium, phosphate, 25(OH) vitamin D, plasma aldosterone concentration (PAC), plasma renin activity (PRA), and aldosterone to renin ratio (ARR) were measured in every patient. Computed tomography (CT) scanning of the adrenal gland and adrenal hormone levels was used to evaluate the function of the adrenal adenoma. We also evaluated the impact of renin-angiotensin-aldosterone system (RAAS) components on PTH from the recumbent-upright test in 15 patients with APA and 30 patients with NFA. RESULTS Compared with NFA, PTH levels were significantly increased in patients with APA, and serum calcium and phosphate were significantly decreased. When position was changed from supine to upright, the variation in PTH levels was significantly higher in APA patients compared with NFA patients. Receiver operator characteristic (ROC) curves identified the Youden index, which corresponded to the best tradeoff of combined marker (ARR and PTH) with a sensitivity and specificity of 89.3% and 93.1%, respectively. CONCLUSIONS The baseline and positional variation of serum PTH levels were significant in APA, thus PTH may be a promising auxiliary index for the clinical diagnosis of APA. Keywords: aldosterone; aldosterone-producing adenoma; blood pressure; hypertension; nonfunctioning adenoma; parathyroid hormone. doi: /ajh/hpv146 Primary aldosteronism (PA) is a heterogeneous group of disorders caused by the secretion of aldosterone, relatively autonomous from the renin-angiotensin-aldosterone system (RAAS). It is the most common cause of secondary hypertension. The incidence of PA among adults with resistant hypertension in China is 7.1%, 1 in which cases aldosterone-producing adenoma (APA) is recognized as the most frequent cause. In recent years, more and more attention is being paid to the nonfunctioning adenoma (NFA) with an increase in the number of incidental finding of so-called incidentalomas. 2 A study in China of 1,173 patients with adrenal incidentaloma showed that 68.97% were nonfunctional tumors. 3 It is difficult to discriminate between APA and NFA based on clinical manifestations, signs, and computed tomography (CT) imaging. Serum hormone levels are additionally required to identify APA. It has been indicated that raised serum parathyroid hormone (PTH) levels are a feature of APA. 4 In vitro studies have shown that PTH increased aldosterone by concentration-dependent pathway in rats. 5 In humans, PTH and PTHrelated peptide (PTH-rP) enhanced aldosterone and cortisol secretion from zona glomerulosa cells, 6 and PTH-rP acts as an autocrine/paracrine factor in the growth and malignancy of adrenocortical tumors. 7 In addition, infusion of aldosterone in rats leads to an increase in PTH levels. 8 It has also been suggested that there is a bidirectional link between the adrenocortical zona glomerulosa and the parathyroid gland. Rossi GP et al. 9 reported on a patient who presented with resistant arterial hypertension and had PA and primary hyperparathyroidism. They revealed the expression of type 1 PTH receptors in aldosterone-producing adrenocortical nodules and mineralocorticoid receptors in the nuclei of parathyroid adenoma cells. Previous studies have demonstrated that PTH levels are higher in APA compared with primary hypertension 4,10,11 and bilateral adrenal hyperplasia 4, Correspondence: Yi-Ming Mu (muyiming@301hospital.com.cn). Initially submitted June 4, 2015; date of first revision June 18, 2015; accepted for publication August 2, 2015; online publication August 24, Medical Center, Tsinghua University, Beijing, China; 2 Department of Endocrinology, Chinese PLA General Hospital, Beijing, China; 3 Medical Research & Biometrics Center, Cardiovascular Institute and Fuwai Hospital, National Center for Cardiovascular Diseases China, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. * These authors contributed equally to this work. American Journal of Hypertension, Ltd All rights reserved. For Permissions, please journals.permissions@oup.com American Journal of Hypertension 29(5) May

2 Zhang et al. and significantly dropped after treatment of PA by either adrenal surgery or mineralocorticoid receptor antagonists 10 accompanied by a remarkable increase in serum calcium and normalized serum K+, plasma aldosterone concentration (PAC), plasma renin activity (PRA), and aldosterone to renin ratio (ARR). 4,10 However, despite changes in calcium implicating a possible aldosterone-induced calciuric effect, studies supported the regulation of PTH levels by the RAAS. Moreover, aldosterone and PTH can contribute to cardiovascular disease 12 and bone loss. 13,14 On the basis of these findings, this study was conducted to explore the interplay between aldosterone and PTH in APA and adrenal NFA. Furthermore, this study aimed to determine an optimal level of serum PTH to distinguish APA from NFA. MATERIALS AND METHODS Patients and measurements Between April 2011 and May 2015, we recruited 154 patients with adrenal adenoma at People Liberation Army General Hospital, of whom 84 were diagnosed as PA due to APA, 58 due to NFA, and 12 were excluded from the current analysis because of the presence of other diseases causing aldosteronism or hyperparathyroidism. We measured the plasma levels of intact PTH, serum Ca2+, Na+, K+, Mg2+, 25(OH) vitamin D, alkaline phosphatase, creatinine, fasting plasma glucose, cholesterol levels, and urinary Na + and K+ excretion in each patient. Echocardiography was conducted to evaluate complications of hypertension. Recumbentupright test, captopril-challenge test, saline-loading test, or upright furosemide-loading test was used to diagnose APA in line with the criteria described by the Japan Endocrine Society. 15 In 15 patients with APA and 30 patients with NFA, we also evaluated serum PTH levels in the supine position for at least 8 hours and after 4 hours of being in the upright position for the recumbent-upright test between September 2014 and May Moreover, 24-hour urinary calcium of them was measured. APA was differentiated from bilateral adrenal hyperplasia by high-resolution CT of the adrenal glands along with selective adrenal vein sampling, and testing of serum adrenocorticotrophin, cortisol, urinary free cortisol, urinary epinephrine, urinary norepinephrine, urinary dopamine, and dexamethasone suppression was also used to exclude Cushing s disease and pheochromocytoma. Serum potassium and sodium levels were measured, in conjunction with a normal sodium diet, using the Hitachi 7600 Automated analyzer (HITACHI Corporation, Tokyo, Japan). Serum and urinary aldosterone was determined by a radioimmunoassay kit (Northern Biotech, Beijing, China) with a detection range of pmol/l, sensitivity of 55 pmol/l, intrabatch coefficient of variation < 10% and interbatch coefficient of variation < 15%. PRA was measured as the generation of angiotensin I in vitro with a radioimmunoassay kit (Northern Biotech, Beijing, China). Serum intact PTH (1 84) was detected by electrochemiluminescence immunoassay (Roche, Mannheim, Germany; normal values, pg/ml). Plasma adrenocorticotrophin level, plasma cortisol, and urinary free cortisol levels were assessed with radioimmunoassay (RIA) kits (Siemens, Marburg, Germany). Testing for 24-hour urinary epinephrine, norepinephrine, and dopamine excretion was regularly performed at People Liberation Army General Hospital using high-pressure liquid chromatography. For CT of the adrenal glands, 3-mm slices were used. Diagnostic criteria The diagnostic criteria of APA included: (i) adrenal adenoma; (ii) hypertension with or without persistent hypokalemia; (iii) an ARR >20 ng/dl per ng/ml/hour (if the ARR was not available because the PRA levels were below the detection limit of the assay, the lowest value would be used as the PRA level); and (iv) at least 1 confirmatory test supporting the diagnosis of APA, including the captopril test, saline infusion test, or furosemide challenge test. Moreover, APA was differentiated from bilateral adrenal hyperplasia by high-resolution CT of the adrenal glands along with selective adrenal vein sampling. And all the patients underwent follow-up after adrenalectomy, confirming the diagnosis of APA. The diagnostic criteria of the nonfunctional adenoma were: (i) adrenal adenoma; (ii) failing to meet the diagnostic criteria of PA (described above); (iii) not diagnosed as Cushing s disease, pheochromocytoma, or other functional adrenal adenoma after systematical measurement of plasma metanephrines and cortisol by screening tests and confirmatory tests. Those patients who had other diseases causing aldosteronism or hyperparathyroidism were excluded. Statistical analysis SPSS software (version 20.0, IBM, Armonk, NY) was used for data analysis. Comparison of continuous variables between the different groups was performed by an independent-samples t-test. Categorical variables were compared by χ 2 test. In all patients, logistic regression analysis was used to assess the correlation between the diagnosis of APA and NFA, with age, gender, body mass index, systolic blood pressure, diastolic blood pressure, serum calcium, serum phosphate, 25(OH) vitamin D, tumor size, and PTH as the independent variables. The results were expressed by mean ± SD or median and range, as appropriate. ARR*PTH was considered as a new combined maker. The diagnostic accuracy of serum PTH, ARR, and ARR*PTH were analyzed by the receiver operator characteristic (ROC) curves. A P value of <0.05 was considered to be significant. RESULTS Baseline characteristics of the patients with APA or NFA are shown in Table 1. Compared with NFA, PTH levels were significantly elevated, and serum potassium, total calcium, phosphate, and ionized calcium were significantly decreased in the patients with APA. Urinary K+ secretion significantly differed between the 2 groups. However, there was no significant difference in serum sodium, magnesium, creatinine, alkaline phosphatase, and 25(OH) vitamin D between the 2 groups. We adjusted for age, gender, BMI, systolic blood 576 American Journal of Hypertension 29(5) May 2016

3 PTH for the Clinical Diagnosis of APA Table 1. Baseline characteristics of the patients with APA or NFA APA (n = 84) NFA (n = 58) P value Age (years) 50 ± ± Sex distribution (female/male) 40/44 37/ BMI (kg/m 2 ) 26.0 ± ± Pulse rate (bpm) 78 ± 8 76 ± SBP (mm Hg) 149 ± ± DBP (mm Hg) 93 ± ± Serum potassium (mmol/l) 2.96 ± ± Serum sodium (mmol/l) ± ± Serum magnesium (mmol/l) 0.88 ± ± Serum calcium (mmol/l) 2.20 ± ± Serum phosphate (mmol/l) 1.04 ± ± Serum ionized calcium (mmol/l) 1.10 ± ± Serum alkaline phosphatase (U/l) 68.9 ± ± Creatinine (mmol/l) 71.1 ± ± Fasting plasma glucose (mmol/l) 5.1 ± ± Total cholesterol (mmol/l) 4.31 ± ± Triacylglycerol (mmol/l) 1.49 ± ± HDL cholesterol (mmol/l) 1.14 ± ± LDL cholesterol (mmol/l) 2.69 ± ± Urinary Na+ excretion (mmol/24 h) ± ± Urinary K+ excretion (mmol/24 h) 56.3 ± ± (OH) vitamin D (ng/ml) 11.5 ± ± PTH (pg/ml) 77.6 ± ± a Abbreviations: APA, aldosterone-producing adenoma; NFA, nonfunctioning adenoma; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; PTH, parathyroid hormone; HDL, high-density lipoprotein; LDL, low-density lipoprotein. a A significant difference between APA and NFA after adjusting for age, gender, BMI, SBP, DBP, serum calcium, serum phosphate, 25(OH) vitamin D, and tumor size. pressure, diastolic blood pressure, serum calcium, serum phosphate, 25(OH) vitamin D, and tumor size, but PTH levels remained significantly increased in APA in contrast to NFA (P = 0.000). As expected, there was no significant difference in tumor size, cortisol, adrenocorticotrophin, urinary free cortisol, urinary epinephrine, urinary norepinephrine, and urinary dopamine, whereas there was marked difference in PRA, PAC, ARR, and urinary aldosterone between the patients with APA and NFA (Table 2). As shown in Table 3, conventional echocardiographic parameters showed significant differences between the 2 groups. Aortic root dimension, left atrial diameter, left ventricular end-diastolic diameter, interventricular septum thickness, posterior wall, right atrial diameter, main pulmonary artery diameter, and the main left ventricular mass index were significantly higher among patients with APA than those with NFA. Figure 1A lists the variation in PTH levels (ΔPTH) resulting from the positional changes in the recumbent- upright test in 45 patients (15 APA and 30 NFA). Compared with patients who had NFA, ΔPTH was significantly higher in the patients with APA (APA = ± 35.67; NFA = ± 12.45; P = 0.003). Figure 1B shows no significant difference in urinary excretion of calcium between patients with APA and NFA (6.76 ± 3.23 vs ± 1.95; P = 0.076). We evaluated the RAAS system and PTH levels at both supine and upright positions. There were significant differences in PRA, PAC, and PTH levels between the different positions both in the patients with APA and NFA (Table 4). The corresponding ROC curves of serum PTH, ARR, and ARR*PTH to distinguish APA from NFA are shown in Figure 2. For the differentiation between APA and NFA, PTH and ARR acted almost equally (P = for comparison), with a slight insignificant advantage for the ARR. However, area under the curve (AUC) of the combined marker (ARR*PTH) was 0.956, which was higher than that of ARR (AUC = 0.938). For all the ROC curves, the dashed line and square dot identified the Youden index, which corresponded with the value providing the best tradeoff of sensitivity and specificity, respectively. The Youden index of PTH was pg/ml, which corresponded to a sensitivity and specificity of 69.0% and 86.2%, respectively. Moreover, American Journal of Hypertension 29(5) May

4 Zhang et al. Table 2. Function evaluation of the adrenal adenoma APA (n = 84) NFA (n = 58) P value Tumor size (cm) 1.6 ± ± Supine PRA (µg/l/h) 0.27 ± ± Supine PAC (pmol/l) ± ± Upright PRA (µg/l/h) 0.62 ± ± Upright PAC (pmol/l) ± ± Upright ARR (pmol/l)/(µg/l/h) ± ± Urinary aldosterone (nmol/24 h) 27.3 ± ± Plasma cortisol (0000 h) (nmol/l) ± ± Plasma cortisol (0800 h) (nmol/l) ± ± Plasma cortisol (1600 h) (nmol/l) ± ± ACTH (0000 h) (pmol/l) 2.64 ± ± ACTH (0800 h) (pmol/l) 8.35 ± ± ACTH (1600 h) (pmol/l) 3.49 ± ± Urinary free cortisol (nmol/24 h) ± ± Urinary epinephrine (μg/24 h) a 49.7 ± ± Urinary norepinephrine (μg/24 h) a ± ± Urinary dopamine (μg/24 h) a ± ± Abbreviations: APA, aldosterone-producing adenoma; NFA, nonfunctioning adenoma; PRA, plasma renin activity; PAC, plasma aldosterone concentration; ARR, aldosterone to renin ratio; ACTH, adrencorticotrophic hormone. a Only 45 patients (APA = 26; NFA = 19) who had been doubt of pheochromocytoma were detected urinary epinephrine, urinary norepinephrine, and urinary dopamine. Table 3. Comparison of conventional echocardiographic variables APA (n = 84) NFA (n = 58) P value AOD (mm) 32.1 ± ± LA (mm) 34.3 ± ± LVEDD (mm) 45.4 ± ± IVS (mm) 11.5 ± ± PW (mm) 10.1 ± ± RA (mm) 33.6 ± ± RVD (mm) 32.2 ± ± MPAD (mm) 23.1 ± ± Ejection fraction (%) 66.6 ± ± Fraction shortening (%) 37.2 ± ± Mean LVMI (g/m 2 ) ± ± Abbreviations: APA, aldosterone-producing adenoma; NFA, nonfunctioning adenoma; AOD, aortic root dimension; LA, left atrial diameter; LVEDD, left ventricular end-diastolic diameter; IVS, interventricular septum thickness; PW, posterior wall; RA, right atrial diameter; RVD, right ventricular dimension; MPAD, main pulmonary artery diameter; LVMI, left ventricular mass index. ROC curves identified the best tradeoff of the combined marker (ARR and PTH) with a sensitivity and specificity of 89.3% and 93.1%, respectively. It was indicated that serum PTH was useful to improve the diagnosis of APA. DISCUSSION In our study, we displayed a biochemically well-characterized cohort of patients with APA and NFA and found that the patients with APA had higher PTH levels and reduced serum calcium and phosphate levels compared to those with NFA. The variation in PTH levels induced by supine-to-upright positional changes was significantly higher in patients with APA when compared to patients with NFA. Of note, we proved that PTH was valuable for the auxiliary diagnosis of APA. Given the evidence of increased PTH in PA, studies describe the interaction between aldosterone and the calcium-regulating hormone system. Serum calcium levels decline in PA 10,13 and rise following treatment, compared with essential hypertension. 11,16 However, urinary calcium in PA was higher in some studies, 13,16 supporting a hypothesis of an influence of aldosteronism on renal calcium handling, resulting in hypercalciuria and consequent hyperparathyroidism. 10,17,18 Although we were in line with findings 4,10 that showed no significant difference in urinary excretion of calcium between patients with APA and NFA (6.76 ± 3.23 vs ± 1.95; P = 0.076), serum calcium, ionized calcium, and phosphate were all significantly decreased and urinary K+ excretion was significantly increased in patients with APA. To some extent, our results support the concept that chronic 578 American Journal of Hypertension 29(5) May 2016

5 PTH for the Clinical Diagnosis of APA Figure 1. ΔPTH levels and urinary excretion of calcium between patients with APA and NFA. (A) The variation in PTH levels (ΔPTH) was significantly higher in APA patients compared with NFA patients, when the position changed from supine to upright. (B) There was no significant difference in urinary excretion of calcium between patients with APA and NFA. Abbreviations: APA, aldosterone-producing adenoma; NFA, nonfunctioning adrenal adenoma; PTH, parathyroid hormone. Table 4. Comparison of RAAS system and PTH levels in recumbent-upright test APA (n = 15) NFA (n = 30) Supine Upright P Supine Upright P PRA (µg/l/h) 0.16 ± ± ± ± PAC (pmol/l) ± ± ± ± PTH (pg/ml) 74.7 ± ± ± ± Abbreviations: APA, aldosterone-producing adenoma; NFA, nonfunctioning adenoma; PRA, plasma renin activity; PAC, plasma aldosterone concentration; PTH, parathyroid hormone. Figure 2. The corresponding receiver operator characteristic curves of serum PTH and ARR to distinguish APA from the patients with NFA. Abbreviations: APA, aldosterone-producing adenoma; NFA, nonfunctioning adrenal adenoma; PTH, parathyroid hormone; AUC, area under the curve; ARR, aldosterone to renin ratio; CI, confidence interval. aldosterone excess induced hyperparathyroidism by the effect of secondary hypercalciuria. However, despite these changes in calcium levels, a direct effect of the RAAS on PTH is also plausible. One study revealed that RAAS inhibitor medications lead to lower PTH levels but no significant change in serum calcium, suggesting that the blockade of the RAAS may have a direct effect on PTH. 19 Though the pathway by which aldosterone or the RAAS stimulates PTH is not clear, research showed that a higher ARR was associated with higher serum PTH concentrations in the general population and thus added to the increasing evidence of a relation between the RAAS and PTH. 20 Our data were in line with these findings and showed for the first time that among patients with APA and NFA, there were significant differences in PRA, PAC, and PTH levels in different positions, and that the variation in PTH levels, when the position was changed from supine to upright, was significantly higher in patients with APA compared to those with NFA. The mechanism that may underlie a significantly higher variation in PTH levels in APA in response to positional changes might involve mutual interaction between RAAS and PTH. Brown et al. 21 studied RAAS components and the regulation of PTH in individuals without PA. From the results, it seemed that the acute modulation of PTH by the RAAS was mediated by angiotensin II, whereas aldosterone may be involved in long-term influences of the RAAS on PTH. In patients with APA, longterm suppression of serum rennin is caused by overproduction of aldosterone, so a slight increase might elevate the concentration of angiotensin II. Another mechanism that has been suggested is that increased protein concentrations in response to assuming the upright position could result in decreased levels of ionized calcium. Aydin et al. 22 found that serum calcium and PTH were affected by positional changes in patients with primary hyperparathyroidism. Because the changes of serum calcium and ionized calcium were not measured in our study, whether PTH changes in accordance with position are regulated by the RAAS system, serum calcium levels, or both of them remain speculative. However, these evidences support that serum PTH levels can be used to evaluate the function of adrenal adenomas. Excess RAAS activity, PTH levels and insufficient vitamin D have all been associated with cardiovascular and skeletal effects. 12,23 In current studies, an increased T-score value of bone mineral density and a higher prevalence of osteoporosis American Journal of Hypertension 29(5) May

6 Zhang et al. was shown in patients with PA compared to patients with essential hypertension. Petramala et al. 14 also found lower 25(OH) vitamin D levels and a higher percentage of vitamin D deficiency in patients with PA, while, our data are in line with the studies that confirm there is no significant changes in serum 25(OH) vitamin D. 13,16 It should be noted that studies have also demonstrated an interaction between vitamin D and RAAS system In our study, the left ventricular end-diastolic diameter, interventricular septum thickness, and posterior wall were significantly thicker in patients with APA than in those with NFA, and patients with APA had a significantly higher left ventricular mass index than those with NFA. However, ejection fraction and fraction shortening were similar in APA and NFA patients. It is well known that hypertension may lead to left ventricular remodeling and function. However, increasing evidence suggests that the potential interplay between PTH and aldosterone might contribute to the pathogenesis of cardiovascular disease. The majority of studies have demonstrated that an excess of PTH levels induced calcium overload and oxidative stress in cardiomyocytes and aggravated the reduction in intra-mitochondrial ATP levels, resulting in subsequent necrotic cell death and myocardial fibrosis. 12,27 However, whether PTH is a determinant of cardiovascular disease remains to be proven by further studies. Recent evidence also focuses on a relevant interaction between klotho and aldosterone, but whether such associations result in cardiovascular disease damage also needs to be clarified. 27 We are the first to find a significant difference in serum PTH levels between APA and NFA in the Chinese population. Both ARR and serum PTH were useful in identifying patients with APA in our population of referred adrenal adenoma patients. Although the AUC of the ROC curve of ARR was higher than that of PTH, the AUC of the combined marker (ARR and PTH) was higher than that of ARR, indicating that serum PTH was useful for the auxiliary diagnosis of APA. The ROC curves indicated that either PTH or PTH combined with ARR had a higher diagnostic value. And it was easy to derive serum PTH levels. Hence, serum PTH levels are an important reference to judge the function of adrenal adenoma. A typical limitation of the present study was the serum PTH deficiency after adrenalectomy. While further investigation is required to evaluate the changes in serum total calcium and ionized calcium in the recumbent-upright test in patients with APA, which would be helpful in clarifying the relationship between calcium-regulatory and adrenalregulatory hormones. Among patients with adrenal adenoma, those with APA displayed a significantly higher baseline and positional variation in serum PTH levels than NFA. 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