Thamara E. Osinga

Catecholamine-Synthesizing Enzymes Are Expressed in Parasympathetic Head and Neck Paraganglioma Tissue

Background/Aim: Increased dopamine production may be a feature of head and neck paraganglioma (HNPGL). 18F-fluorodihydroxyphenylalanine positron emission tomography scintigraphy has a high sensitivity for detecting HNPGLs. These observations strongly suggest that HNPGLs have the capacity for L-3,4-dihydroxyphenylalanine uptake and conversion towards dopamine. Therefore, our aim was to demonstrate the presence of catecholamine-synthesizing enzymes, i.e. tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC) and dopamine β-hydroxylase (DBH) in HNPGL tissue. Methods: A single-center study was performed among patients who underwent surgery for HNPGL at a single university referral center between 1994 and 2012. HNPGL tissue was immunohistochemically stained for TH, AADC and DBH. Data on paraganglioma-associated germline mutations, preoperative biochemical phenotype and imaging studies were retrieved. Catecholamine excess was defined as preoperative plasma and/or urinary levels of metanephrine, normetanephrine or 3-methoxytyramine above the upper reference limit. Results: Nineteen HNPGLs from 18 patients were evaluated. All tumor tissues (100%) stained positive for AADC, 6 (32%) for TH and 2 (11%) for DBH. Of 3 HNPGLs staining positive for DBH, 2 were also positive for AADC and TH. Catecholamine excess was only present in 1 patient (5%). The HNPGLs of this single patient only showed positive staining for AADC. Conclusions: Catecholamine-synthesizing enzymes, in particular AADC, are expressed in the majority of HNPGL tissues.

Unilateral and Bilateral Adrenalectomy for Pheochromocytoma Requires Adjustment of Urinary and Plasma Metanephrine Reference Ranges

CONTEXT Follow-up after adrenalectomy for pheochromocytoma is recommended because of a recurrence risk. During follow-up, plasma and/or urinary metanephrine (MN) and normetanephrine (NMN) are interpreted using reference ranges obtained in healthy subjects. OBJECTIVE Because adrenalectomy may decrease epinephrine production, we compared MN and NMN concentrations in patients after adrenalectomy to concentrations in a healthy reference population. DESIGN A single-center cohort study was performed in pheochromocytoma patients after adrenalectomy between 1980 and 2011. SUBJECTS Seventy patients after unilateral and 24 after bilateral adrenalectomy were included. MAIN OUTCOME MEASURES Plasma-free and urinary-deconjugated MN and NMN determined at 3 to 6 months and annually until 5 years after adrenalectomy were compared with concentrations in a reference population. Data are presented in median (interquartile range). RESULTS Urinary and plasma MN concentrations 3 to 6 months after unilateral adrenalectomy were lower compared with the reference population (39 [31-53] μmol/mol creatinine and 0.14 [0.09-0.18] nmol/L vs 61 [49-74] μmol/mol creatinine and 0.18 [0.13-0.23] nmol/L, respectively, both P < .05). Urinary MN after bilateral adrenalectomy was reduced even further (7 [1-22] μmol/mol creatinine; P < .05). Urinary and plasma NMN were higher after unilateral adrenalectomy (151 [117-189] μmol/mol creatinine and 0.78 [0.59-1.00] nmol/L vs 114 [98-176] μmol/mol creatinine and 0.53 [0.41-0.70] nmol/L; both P < .05). Urinary NMN after bilateral adrenalectomy was higher (177 [106-238] μmol/mol creatinine; P < .05). Changes in urinary and plasma MNs persisted during follow-up. CONCLUSION Concentrations of MN are decreased, whereas NMN concentrations are increased after unilateral and bilateral adrenalectomy. Adjusted reference values for MN and NMN are needed in the postsurgical follow-up of pheochromocytoma patients.

Dopamine concentration in blood platelets is elevated in patients with head and neck paragangliomas

Abstract Background: Plasma 3-methoxytyramine (3-MT), a metabolite of dopamine, is elevated in up to 28% of patients with head and neck paragangliomas (HNPGLs). As free dopamine is incorporated in circulating platelets, we determined dopamine concentration in platelets in patients with a HNPGL. Methods: A single center cohort study was performed between 2012 and 2014. Thirty-six patients with a HNPGL were compared to healthy controls (68 for dopamine in platelets and 120 for plasma 3-MT). Results: Dopamine concentration in platelets was elevated in HNPGL patients compared to healthy controls (median [interquartile ranges] 0.48 [0.32–0.82] pmol/109 platelets vs. 0.31 [0.24–0.47] pmol/109 platelets; p<0.05), whereas plasma 3-MT concentration did not differ between both groups (0.06 [0.06–0.08] nmol/L vs. 0.06 [0.06–0.06] nmol/L; p=0.119). Based on 68 healthy controls, the reference interval for dopamine concentration in platelets was 0.12–0.97 pmol/109 platelets. Six (16.7%) patients with a HNPGL demonstrated an increased dopamine concentration in platelets compared to three (8.3%) patients with an increased plasma 3-MT level (p=0.053). The sensitivity and specificity were 16.7% and 98.5% for platelet dopamine and 8.3% and 97.5% for plasma 3-MT concentration (p=0.37). Conclusions: Dopamine concentration in platelets is elevated in patients with a HNPGL compared to healthy subjects, and may be a novel biomarker for dopamine producing paraganglioma.

No influence of antihypertensive agents on plasma free metanephrines

BACKGROUND Hypertension can be the predominant sign of pheochromocytoma (PCC) and sympathetic paraganglioma (sPGL) and screening for PCC/sPGL is often performed in patients who are already being treated with antihypertensive agents. There is very little information about the influence of antihypertensive drugs on plasma free metanephrines. The aim of this study was to determine whether commonly prescribed antihypertensive drugs can falsely elevate plasma free metanephrines concentrations measured by LC-MS/MS analysis. METHODS In a prospective study we included patients with newly diagnosed hypertension, who started monotherapy with an antihypertensive agent (i.e. β-blocker, thiazide diuretic or angiotensin-converting enzyme (ACE) inhibitor). Plasma free metanephrine (MN) and normetanephrine (NMN) levels were measured before and one month after the start of the medication quantified by LC-MS/MS. RESULTS Between 2009 and 2014, 39 patients were included (β-blocker n=13, thiazide diuretic n=14 and ACE inhibitor n=12). In the whole group, the median plasma free MN and NMN concentrations at baseline were 0.19 [0.17-0.26] nmol/L and 0.56 [0.38-0.95] nmol/L. One month after the start of antihypertensive treatment, the median plasma free MN and NMN concentrations were comparable; 0.20 [0-16-0.24] nmol/L and 0.63 [0.39-0.75] nmol/L, respectively (P=0.43 and P=0.39). Separate analysis for each of the three antihypertensive agents examined did not reveal any significant changes in the median plasma free MN and NMN concentrations. CONCLUSIONS The measurement of plasma free MN and NMN with LC-MS/MS is not affected by use of β-blockers, diuretics and ACE inhibitors. Withdrawal of these drugs prior to the quantification of plasma metanephrines is therefore not necessary.

Emerging role of dopamine in neovascularization of pheochromocytoma and paraganglioma

Dopamine is a catecholamine that acts both as a neurotransmitter and as a hormone, exerting its functions via dopamine (DA) receptors that are present in a broad variety of organs and cells throughout the body. In the circulation, DA is primarily stored in and transported by blood platelets. Recently, the important contribution of DA in the regulation of angiogenesis has been recognized. In vitro and in vivo studies have shown that DA inhibits angiogenesis through activation of the DA receptor type 2. Overproduction of catecholamines is the biochemical hallmark of pheochromocytoma (PCC) and paraganglioma (PGL). The increased production of DA has been shown to be an independent predictor of malignancy in these tumors. The precise relationship underlying the association between DA production and PCC and PGL behavior needs further clarification. Herein, we review the biochemical and physiologic aspects of DA with a focus on its relations with VEGF and hypoxia inducible factor related angiogenesis pathways, with special emphasis on DA producing PCC and PGL.—Osinga, T. E., Links, T. P., Dullaart, R. P. F., Pacak, K., van der Horst‐Schrivers, A. N. A., Kerstens, M. N., Kema, I. P. Emerging role of dopamine in neovascularization of pheochromocytoma and paraganglioma. FASEB J. 31, 2226–2240 (2017). www.fasebj.org

SDH Subunit Mutation Status in Saliva: Genetic Testing in Patients with Pheochromocytoma

Germline mutations occur in up to 30-40% of pheochromocytoma/paraganglioma, with mutations in the succinate dehydrogenase (SDH) subunits B (SDHB) and D (SDHD) being the most common. Blood samples are favored for obtaining high quality DNA, however, leukocytes can also be obtained by collecting saliva. The aim of this study was to determine whether SDHB and SDHD gene mutations in patients with pheochromocytoma/paraganglioma could be determined using a salivary sample. Paired blood and salivary samples were collected from 30 patients: 9 SDHB mutation positive, 13 with a SDHD mutation, and 8 without any SDHx mutations. The Oragene DISCOVER kit was used to collect and extract DNA from saliva. Blood DNA was extracted from EDTA blood samples. The DNA purification and concentration were measured by spectrophotometry. The 8 exons of SDHB and the 4 exons of SDHD were amplified and sequenced by PCR-based bidirectional Sanger sequencing. Total DNA yields from blood DNA were similar to those obtained from saliva DNA [mean (±SD) saliva vs. blood DNA concentration 514.6 (±580.8) ng/µl vs. 360.9 (±262.7) ng/µl; p=0.2)]. The purity of the saliva DNA samples was lower than that of blood [mean OD260/OD280 ratio 1.78 (±0.13) vs. 1.87 (±0.04); p=0.001, respectively], indicating more protein contamination in the saliva-extracted DNA. This study shows that salivary DNA collected from patients with pheochromocytoma/paraganglioma is a good alternative for extraction of genomic DNA for its high DNA concentration and acceptable purity and can be used as an alternative to blood derived DNA in screening for SDHB and SDHD mutations.

Mass spectrometric quantification of salivary metanephrines-A study in healthy subjects

BACKGROUND Determination of metanephrine (MN), normetanephrine (NMN), and 3-methoxytyramine (3-MT) in saliva may offer potential diagnostic advantages in diagnosing pheochromocytoma. METHODS In this preliminary study, we determined metanephrine concentrations in saliva of healthy subjects and the relationship with simultaneously measured plasma metanephrines. We also studied the possible influence of pre-analytical conditions such as a collection device, awakening, posture, and eating on the salivary metanephrine levels. RESULTS Eleven healthy subjects were included. Fasting blood and saliva samples were collected in seated position and after 30min of horizontal rest. Plasma and salivary MN, NMN, and 3-MT concentrations were determined using a high-performance liquid chromatography tandem mass spectrometric technique (LC-MS/MS) with automated solid phase extraction sample preparation. Metanephrines were detectable in saliva from all participants both in seated and supine position. No significant correlations were observed between the MN, NMN, and 3-MT concentrations in saliva and plasma in seated or supine position. Furthermore, there was no difference between MN, NMN, and 3-MT samples collected with or without a collection device. CONCLUSION Metanephrines can be detected in saliva with LC-MS/MS with sufficient sensitivity and precision. Our findings warrant evaluation of salivary metanephrine measurement as a novel laboratory tool in the work-up of patients suspected of having a pheochromocytoma.

Contents Vol. 101, 2015

I.J. Clarke, Clayton, Vic. C. Coen, London M.A. Cowley, Clayton, Vic. W.W. de Herder, Rotterdam S.L. Dickson, Gothenburg A. Enjalbert, Marseille A.B. Grossman, Headington D. Jezová, Bratislava A.S. Kauff man, La Jolla, Calif. M.J. Kelly, Portland, Oreg. P.A. Kelly, Paris X. Ni, Shanghai S.R. Ojeda, Beaverton, Oreg. (Reviews) G. Rindi, Rome H. Sasano, Sendai J.Y. Seong, Seoul M. Tena-Sempere, Cordoba H. Tsukamura, Nagoya E.J. Wagner, Pomona, Calif. B. Wiedenmann, Berlin Z. Zeng, Beijing Editorial Board