Evaluation of bone mineral density (BMD) and trabecular bone score (TBS) in pheochromocytoma and paraganglioma; a multi-centric case-control study from India.

Pheochromocytoma and paraganglioma (PPGL) have been associated with low bone mineral density (BMD) due to excess sympathetic system stimulation. Our study revealed low BMD and TBS (trabecular bone score) in cases compared to matched controls. Plasma-free nor-metanephrine and hypertension duration found to be most consistent predictive factors. PURPOSE: Pheochromocytoma and paraganglioma (PPGL) have been associated with low bone mineral density (BMD) and increased fracture risks. Sympathetic nervous system stimulation has been shown to increase bone resorption and decrease bone formation via ß2 receptors. Chronic inflammation and increased cytokine production add to more bone loss. TBS (trabecular bone score) is an established surrogate marker for bone histomorphometry. BMD and TBS data in pheochromocytoma and PPGL are scarce. The aim was to assess the BMD and TBS in pheochromocytoma and PPGL and look for clinical and biochemical predictors. METHODS: This case-control study had sample size of 58 (29 cases and controls each). BMI-, age-, and sex-matched controls were taken for comparison. Both cases and controls had undergone DXA scan and BMD {Z-scores and bone mineral concentration (BMC) in g/cm2} and TBS were analyzed. Detailed clinical histories and relevant biochemistry values were noted. RESULTS: The mean age of our case population was 29.5 ± 9.4 years with a mean age of HTN onset at 26.86 ± 6.6 years. Lumbar spine BMC (0.86 ± 0.14 vs 0.96 ± 0.15; p = 0.036), femoral neck Z-score (- 1.23 ± 1.07 vs - 0.75 ± 0.97; p = 0.003), and whole body BMC (0.91 ± 0.14 vs 1.07 ± 0.11; p = 0.000) were significantly low in cases compared to controls. Similarly, TBS was significantly lower in cases compared to controls (1.306 ± 0.113 vs 1.376 ± 0.083; p = 0.001). CONCLUSION: This study establishes both low bone mass and poor bone quality in an Indian pheochromocytoma and PPGL patient's cohort. Plasma-free nor-metanephrine and duration of hypertension were found to be most consistent predictive factors in multivariate regression analysis.

Giant Cystic Pheochromocytoma Associated With Neurofibromatosis Type 1: A Case Report.

Pheochromocytomas are tumors that develop from the chromaffin cells of the adrenal medulla. More than 40% of cases of pheochromocytomas are associated with genetic conditions such as neurofibromatosis type 1 (NF1) or von Hippel-Lindau syndrome. Cystic pheochromocytomas are rare, generally asymptomatic, and thus of bigger size at the time of diagnosis. Surgical treatment is necessary to prevent cardiovascular morbidity and malignancy risk. We report the case of a 27-year-old patient admitted for further examination of a left adrenal mass that was discovered by an abdominal CT scan in the context of abdominal pain associated with hypertension evolving for three years. The clinical examination showed the presence of multiple café au lait spots, axillary and inguinal freckling with two dermal neurofibromas diagnosed clinically, as well as Lisch nodules on bilateral ophthalmic examination, thus meeting the clinical criteria for the diagnosis of NF1. The clinical laboratory investigation showed elevated urinary metanephrine and normetanephrine levels. CT scan examination showed a 10 cm left adrenal cystic mass on abdominal CT. This mass uptake of the radioligand in metaiodobenzylguanidine (MIBG) scintigraphy without secondary extra-adrenal localization allowed the diagnosis of a seemingly benign cystic pheochromocytoma to be made. The patient was put on presurgical drug preparation with volume expansion and then underwent left unilateral adrenalectomy. The histopathological study was in favor of a rather aggressive cystic pheochromocytoma with a pheochromocytoma of the adrenal gland scaled (PASS) score of 9. Blood pressure and urine catecholamines at seven days, three months, six months, and one year after surgery were normalized. Cystic pheochromocytoma is a rare tumor with a potentially poor prognosis. It is characterized by a more insidious evolution and a larger volume at diagnosis. It should be considered a diagnosis in patients with a cystic adrenal mass or an extra-adrenal mass with fluctuating blood pressure during surgery. This case illustrates the importance of both presurgical preparation and screening for pheochromocytoma in neurofibromatosis type 1.

A Case Report and Literature Review of Pheochromocytoma Without Tachycardia.

This case report highlights a 47-year-old woman with an adrenal incidentaloma and a history of polysubstance abuse, finally diagnosed with pheochromocytoma. Characterized by episodic hypertension, headaches, and palpitations, pheochromocytoma is a rare condition with potential complications like uncontrolled hypertension and heart failure. Remarkably, during her 28-day hospitalization, continuous monitoring revealed no instances of tachycardia or arrhythmias despite multiple symptomatic episodes. This finding aligns with reports that while 50-70% of symptomatic pheochromocytoma patients experience palpitations, only about 20% exhibit detectable tachycardia or arrhythmias. This discrepancy suggests varied individual cardiovascular responses to catecholamine surges, possibly due to differences in catecholamine inactivation rates and receptor sensitivity. This case underscores the complexity of pheochromocytoma symptoms and highlights the need for personalized diagnostic and management strategies. Furthermore, it points to a significant gap in understanding the correlation between palpitations and arrhythmia in pheochromocytoma, indicating a critical area for future research.

Clinical Significance of Pheochromocytoma Size on the Timing and Extent of Surgery.

INTRODUCTION: Elevated metanephrine and catecholamine levels 3-fold upper limit of normal (ULN) are diagnostic for pheochromocytoma. We sought to determine whether size correlates with biochemical activity or symptoms which could guide timing of surgery. METHODS: Data from consecutive patients undergoing adrenalectomy for pheochromocytoma at our institution over a 10-year period were retrospectively collected. These included maximal lesion diameter on preoperative imaging, plasma/urine metanephrine and/or catecholamine levels, demographic variables and presence of typical paroxysmal symptoms. Receiver operating characteristic curves were used to assess predictive accuracy. RESULTS: Sixty-three patients were included in the analysis (41 females and 22 males). Median age was 56 (43, 69) years. Due to various referring practices, 31 patients had documented 24-h urine metanephrine, 26 had 24-h urine catecholamine, and 52 had fractionated plasma metanephrine levels available for review. Values were converted to fold change compared to ULN and the maximum of all measured values was used for logistic regression. Median tumor size was 3.40 (2.25, 4.55) cm in greatest dimension. Tumor size at which pheochromocytoma produced > 3-fold ULN was ≥2.3 cm (AUC of 0.84). Biochemical activity increased with doubling tumor size (odds ratio = 8, P = 0.0004) or ≥ 1 cm increase in tumor size (odds ratio = 3.03, P = 0.001). 40 patients had paroxysmal symptoms, but there was no significant correlation between tumor size/biochemical activity and symptoms. CONCLUSIONS: In our study, tumor size directly correlated with the degree of biochemical activity and pheochromocytomas ≥2.3 cm produced levels 3 times ULN. These findings may allow clinicians to adjust timing of operative intervention.

False-positive results for pheochromocytoma associated with norepinephrine reuptake blockade.

Measurements of plasma metanephrines and methoxytyramine provide a sensitive test for diagnosis of pheochromocytoma/paraganglioma. False-positive results remain a problem, particularly in patients taking norepinephrine reuptake-blocking drugs. Therefore, in this retrospective observational study, we measured plasma metanephrines and methoxytyramine in 61 patients taking norepinephrine reuptake blockers (tricyclic antidepressants or serotonin-norepinephrine reuptake inhibitors) and 17 others taking selective serotonin reuptake inhibitors, all without pheochromocytoma/paraganglioma. We highlight a singular case with strongly elevated plasma normetanephrine and methoxytyramine concentrations associated with norepinephrine reuptake blockade. Data were compared to results from 252 and 1804 respective patients with and without tumors. Plasma normetanephrine was 40% higher (P < 0.0001) in patients on norepinephrine reuptake blockers and methoxytyramine was 127% higher (P = 0.0062) in patients taking tricyclic antidepressants compared to patients not taking uptake blockers and without tumors. The corresponding false-positive rates rose (P < 0.0001) from 4.8% to 23.0% for normetanephrine and from 0.9% to 28.6% for methoxytyramine. Selective serotonin reuptake inhibitors did not increase plasma concentrations of metabolites. In the highlighted case, plasma normetanephrine and methoxytyramine were elevated more than six times above upper reference limits. A pheochromocytoma/paraganglioma, however, was excluded by functional imaging. All biochemical test results normalized after discontinuation of norepinephrine reuptake blockers. These findings clarify that norepinephrine reuptake blockers usually result in mild elevations of normetanephrine and methoxytyramine that, nevertheless, significantly increase the number of false-positive results. There can, however, be exceptions where increases in normetanephrine and methoxytyramine reach pathological levels. Such exceptions may reflect failure of centrally mediated sympathoinhibition that normally occurs with the norepinephrine reuptake blockade.

Comparison of Perioperative Outcomes Between Laparoscopic and Robot-Assisted Adrenalectomy for Large Pheochromocytoma (≥ 5cm): A Retrospective Study.

Purpose: The objective of this study was to compare perioperative outcomes in patients with large (≥5cm) pheochromocytomas who underwent adrenalectomy. Patients and Methods: We retrospectively reviewed patients who underwent laparoscopic adrenalectomy (LA) and robot-assisted adrenalectomy (RA) for large pheochromocytoma (≥ 5cm) at our center between January 2015 to February 2023. We compared the perioperative outcomes between the two groups and investigated impact of high Nor-Metanephrine (NMN) levels on perioperative outcomes by analyzing this subgroup. Results: A total of 115 patients were included in the study, with 48 patients in the robotic group and 67 patients in the laparoscopic group. The following significant difference were identified in favor of RA: shorter operative (excluding docking time) time (190.0 vs 220.0 min, p=0.002), lower estimated blood loss (50.0 vs 120.0 mL, p=0.013), however, RA group has higher surgical expenses (37933.0 vs 7936.0 CNY, p< 0.001). This finding remained consistent when analyzing patients with high NMN levels. Conclusion: Patients with large pheochromocytoma may experience reduced blood loss and shorter operative time when undergoing robot-assisted adrenalectomy. However, it is important to note that the RA approach is associated with significantly higher costs.

A case report and literature review: pheochromocytoma-mediated takotsubo cardiomyopathy, which is similar to acute myocardial infarction.

A 52-year-old Chinese woman was admitted to a cardiac intensive care unit (CCU) due to nausea, vomiting, and dyspnea, which began a day before her hospitalization. Metoprolol succinate and conventional treatment for acute myocardial infarction (AMI) were initially administered to the patient based on electrocardiogram (ECG) findings and elevated cardiac troponin I (cTnI). However, the following day, she developed aggravated nausea, vomiting, fever, sweating, a flushed face, a rapid heart rate, and a significant rise in blood pressure. Furthermore, ultrasonic cardiography (UCG) displayed takotsubo-like changes; nevertheless, ECG indicated inconsistent cTnI peaks with extensive infarction. After coronary computed tomography angiography (CTA) ruled out (AMI), and in conjunction with the uncommon findings, we strongly suspected that the patient had a secondary condition of pheochromocytoma-induced takotsubo cardiomyopathy (Pheo-TCM). In the meanwhile, the use of metoprolol succinate was promptly discontinued. This hypothesis was further supported by the subsequent plasma elevation of multiple catecholamines and contrast-enhanced computed tomography (CECT). After one month of treatment with high-dose Phenoxybenzamine in combination with metoprolol succinate, the patient met the criteria for surgical excision and successfully underwent the procedure. This case report demonstrated that pheochromocytoma could induce TCM and emphasized the significance of distinguishing it from AMI (in the context of beta-blocker usage and anticoagulant management).

Plasma-free metanephrines, nerve growth factor, and renalase significance in patients with PCOS.

PURPOSE: Polycystic ovarian syndrome (PCOS) is a common heterogeneous condition with probably multifactorial genesis. Animal studies have proven the essential role of the sympathetic nervous system in the syndrome development, while human studies are still contradictory. The present study aims to investigate the possible influence of plasma-free metanephrine (MN), and normetanephrine (NMN), nerve growth factor (NGF), and renalase (RNL) on the hormonal and metabolic parameters in women with PCOS and healthy controls. METHODS: Fifty patients with PCOS and 30 healthy women participated in the study. The plasma-free MN and NMN, NGF, RNL, anti-Mullerian hormone (AMH), gonadotropin, androgen levels, and metabolic parameters were investigated. RESULTS: Plasma-free NMN and NGF concentrations were increased in PCOS individuals, while RNL levels were decreased compared to healthy volunteers. Increased plasma-free NMN (OR = 1.0213 [95%CI 1.0064-1.0364], p = 0.005) and NGF (OR = 1.0078 [95%CI 1.0001-1.0155], p = 0.046) but not MN or RNL levels were associated with a higher risk of PCOS after adjustment for age. Plasma-free NMN levels were positively associated with the LH (r = +0.253; p = 0.039). androstenedione (r = +0.265; p = 0.029), 17-OH progesterone (r = +0.285; p = 0.024), NGF (r = +0.320; p = 0.008), and AMH (r = +0.417; p < 0.001) concentrations of the investigated women. RNL levels were inversely related to the BMI (r = -0.245; p = 0.029), HOMA-IR (r = -0.250; p = 0.030), free testosterone (r = -0.303; p = 0.006) levels. systolic (r = -0.294; p = 0.008) and diastolic (r = -0.342; p = 0.002) blood pressure. CONCLUSIONS: Increased sympathetic noradrenergic activity and NGF synthesis might be related to the increased AMH and delta-4 androgen levels in a subgroup of PCOS patients. RNL levels might influence the metabolic status of PCOS patients. Further studies are needed to explore the significance of adrenal medullar and autonomic dysfunction for developing different PCOS phenotypes and their subsequent cardiovascular complications.

Reference intervals for plasma, urinary, and salivary concentrations of free metanephrines in dogs: Relevance to the diagnosis of pheochromocytoma.

BACKGROUND: Measurement of free metanephrines is recommended for screening of pheochromocytoma (PCC) but requires appropriate reference intervals (RIs). HYPOTHESIS/OBJECTIVES: To report RIs for plasma, urinary and salivary concentrations of free metanephrines and to determine the diagnostic performance of plasma free normetanephrine (pNMN) and metanephrine (pMN) concentrations in dogs with PCC, hypercortisolism (HC), and nonadrenal illness (NAI). ANIMALS: Eighty healthy dogs, 11 PCC dogs, 25 HC dogs, 6 NAI dogs. METHODS: Plasma, urine, and saliva were collected prospectively from healthy dogs, and free metanephrine concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, medical records of dogs that had plasma free metanephrine concentrations measured by LC-MS/MS between 2018-2021 were studied retrospectively. RESULTS: The RIs for free metanephrines in plasma, urine and saliva are reported. Dogs with PCC had significantly higher pNMN than dogs with HC (P < .001) and NAI (P = .002). The PCC dogs had significantly higher pMN than HC dogs (P < .001), but not higher than NAI dogs (P = .29). Using the upper reference limit, pNMN (>3.56 nmol/L) showed high sensitivity (100%, 95% confidence interval [CI]: 72-100) and specificity (94%, 95% CI: 79-99) for diagnosis of PCC, whereas pMN (>2.49 nmol/L) showed moderate sensitivity (73%, 95% CI: 39-94) and high specificity (94%, 95% CI: 79-99). CONCLUSIONS AND CLINICAL IMPORTANCE: With establishment of these RIs, biochemical testing for PCC in dogs can be substantially improved. Measurement of pNMN is superior to pMN in dogs with PCC.

Preoperative Factors Associated with Intraoperative Maximum Arterial Pressures in Patients with Pheochromocytoma and Paraganglioma.

Background: Surgery for pheochromocytoma and paraganglioma (PPGL) can lead to life-threatening complications, such as intraoperative hypertensive crises, even when adequate doses of preoperative α-receptor blockades are administered. Objectives: The aim of this study was to identify preoperative factors associated with intraoperative maximum arterial pressure (AP) in patients with PPGL. Methods: We retrospectively reviewed the cases of 61 PPGL patients who underwent surgical resection in our hospital between 2006 and 2020. The primary outcome was intraoperative maximum AP as a single index for continuous variables. Simple and multiple linear regression model were used for statistical analysis. Results: The median maximum systolic AP during surgery was 165 mmHg (interquartile range: 150 - 180 mmHg). Log24-h urinary-fractionated metanephrine (MN) and normetanephrine (NMN) was correlated with intraoperative maximum AP (R-squared = 0.218, P < 0.001). Multiple regression analyses showed that diabetes mellitus, one or more of the classic triad, and log24-h urinary-fractionated MN and NMN were independent factors associated with intraoperative maximum AP. Conclusions: Patients with PPGL accompanied by diabetes mellitus, one or more of the classic triad, and high log 24-h urinary-fractionated MN and NMN values may be at risk for hypertensive crises during surgery regardless of whether preoperative α-receptor blockades are used. Clinicians should manage these patients more carefully and effectively.

Metanephrine and normetanephrine associated with subclinical myocardial injuries in pheochromocytoma and paraganglioma.

Objective: To analyze the correlation between metanephrine and normetanephrines (MNs) and subclinical myocardial injuries (SMI) diagnosed by low left ventricular global longitudinal strain (LV GLS) in patients with pheochromocytoma and paraganglioma (PPGL). Methods: Seventy-six patients who underwent surgery for pheochromocytoma or paraganglioma from September 2017 to April 2022 were examined. All the patients enrolled had normal left ventricular ejection fraction (LVEF) and myocardial injury biomarkers including cardiac troponin I and B-natriuretic peptide. Univariate analysis and multivariate analysis were performed to evaluate the association of MNs and subclinical myocardial injury (SMI)(defined as LV GLS<18). Results: LV GLS of 13(17.11%) PPGL patients was less than 18. The percentage of patients with elevation of single hormone (metanephrine, normetanephrine, 3-methoxytyramine) or any one of MNs was not significantly correlated with SMI (P=0.987, 0.666, 0.128 and 0.918, respectively). All MNs elevation was associated with SMI (OR: 11.27; 95% CI, 0.94-135.24; P= 0.056). After adjusting for age, All MNs elevation was significantly correlated with SMI (OR: 16.54; 95% CI, 1.22-223.62; P= 0.035). Conclusion: MNs might be an important factor influencing myocardial function. All MNs elevation might indicate SMI. If all MNs elevated, LV GLS measurement was recommended for PPGL patients to detect SMI in the absence of decrease LVEF or other heart disease in clinical practice.

Noradrenergic Pheochromocytoma: A Case Report.

Pheochromocytomas and paragangliomas are rare neuroendocrine tumors developed from chromaffin cells. They are exceptional in children with an atypical symptomatology. We report here a case of a 17-year-old boy who presented with a left retroperitoneal mass discovered on thoracic-abdominal-pelvic computed tomography (CT) scan in the presence of diffuse abdominal pain, more pronounced in the left hypochondrium. The exploration had objectified an exclusive secretion of urinary normetanephrine over 24 hours. Metanephrine and 3 ortho methyldopamine were within normal limits. He had adrenalectomy after controlling his blood pressure with an alpha blocker. Histology had confirmed a pheochromocytoma of non-aggressive potential Pheochromocytoma of the Adrenal gland Scaled Score (PASS) 2. The evolution was favorable with normalization of blood pressure and urinary catecholamines at one week, three months, six months, and one year. He tested negative for hereditary syndromic including Von Hippel-Lindau (VHL), RET genes, subject to the succinate dehydrogenase complex B and D subunit genes (SDHB-D), which have been requested.

Preanalytical Considerations and Outpatient Versus Inpatient Tests of Plasma Metanephrines to Diagnose Pheochromocytoma.

CONTEXT: Sampling of blood in the supine position for diagnosis of pheochromocytoma and paraganglioma (PPGL) results in lower rates of false positives for plasma normetanephrine than seated sampling. It is unclear how inpatient vs outpatient testing and other preanalytical factors impact false positives. OBJECTIVE: We aimed to identify preanalytical precautions to minimize false-positive results for plasma metanephrines. METHODS: Impacts of different blood sampling conditions on plasma metanephrines were evaluated, including outpatient vs inpatient testing, sampling of blood in semi- vs fully recumbent positions, use of cannulae vs direct venipuncture, and differences in outside temperature. A total of 3147 patients at 10 tertiary referral centers were tested for PPGL, including 278 with and 2869 without tumors. Rates of false-positive results were analyzed. RESULTS: Outpatient rather than inpatient sampling resulted in 44% higher plasma concentrations and a 3.4-fold increase in false-positive results for normetanephrine. Low temperature, a semi-recumbent position, and direct venipuncture also resulted in significantly higher plasma concentrations and rates of false-positive results for plasma normetanephrine than alternative sampling conditions, although with less impact than outpatient sampling. Higher concentrations and rates of false-positive results for plasma normetanephrine with low compared with warm temperatures were only apparent for outpatient sampling. Preanalytical factors were without impact on plasma metanephrines in patients with PPGL. CONCLUSION: Although inpatient blood sampling is largely impractical for screening patients with suspected PPGL, other preanalytical precautions (eg, cannulae, warm testing conditions) may be useful. Inpatient sampling may be reserved for follow-up of patients with difficult to distinguish true- from false-positive results.

Dual-Template Magnetic Molecularly Imprinted Polymer for Simultaneous Determination of Spot Urine Metanephrines and 3-Methoxytyramine for the Diagnosis of Pheochromocytomas and Paragangliomas.

A novel dual-template magnetic molecularly imprinted polymer (MMIP) was synthesized to extract normetanephrine (NMN), metanephrine (MN) and 3-methoxytyramine (3-MT) from spot urine samples. As the adsorbent of dispersive solid-phase extraction (d-SPE), the MMIP was prepared using dopamine and MN as dual templates, methacrylic acid as the functional monomer, ethylene glycol dimethacrylate as the crosslinking reagent and magnetic nanoparticles as the magnetic core. NMN, MN, 3-MT and creatinine (Cr) in spot urine samples were selectively enriched by d-SPE and detected by HPLC-fluorescence detection/ultraviolet detection. The peak area (A) ratios of NMN, MN and 3-MT to Cr were used for the diagnosis of pheochromocytomas and paragangliomas (PPGLs). The results showed that the adsorption efficiencies of MMIP for target analytes were all higher than 89.0%, and the coefficient variation precisions of intra-assay and inter-assay for the analytes were within 4.9% and 6.3%, respectively. The recoveries of the analytes were from 93.2% to 112.8%. The MMIP was still functional within 14 days and could be reused at least seven times. The d-SPE and recommended solid-phase extraction (SPE) were both used to pretreat spot urine samples from 18 PPGLs patients and 22 healthy controls. The correlation coefficients of ANMN/ACr and AMN/ACr between d-SPE and SPE were both higher than 0.95. In addition, the areas under the receiver operator curves for spot urine ANMN/ACr, AMN/ACr and plasma free NMN and MN were 0.975, 0.773 and 0.990, 0.821, respectively, indicating the two methods had the similar performances. The d-SPE method took only 20 min, which was effective in clinical application.

Clonidine suppression test for a reliable diagnosis of pheochromocytoma: When to use.

OBJECTIVE: In clinical practice, false-positive results in biochemical testing for suspected pheochromocytoma/paraganglioma (PPGL) are not infrequent and may lead to unnecessary examinations. We aimed to evaluate the role of the clonidine suppression test (CST) in the era of analyses of plasma-free metanephrines for the diagnosis or exclusion of PPGL in patients with adrenal tumours and/or arterial hypertension. DESIGN AND METHODS: This single-centre, prospective trial investigated the use of CST in 60 patients with suspected PPGL associated with out-patient elevations of plasma normetanephrine (NMN) and/or metanephrine (MN), in most cases accompanied with hypertension or an adrenal mass. Measurements of plasma catecholamines and free metanephrines were performed by liquid chromatography with electrochemical detection and tandem mass spectrometry, respectively. RESULTS: Forty-six patients entered final analysis (n = 20 with PPGL and n = 26 with a nonfunctional adrenal mass and/or hypertension). CST reliably excluded false-positive baseline NMN results with a specificity of 100%. The sensitivity of CST improved from 85% to 94% when tumours with isolated MN increase (n = 3) were not considered. In patients with elevated baseline NMN (n = 24), CST correctly identified all patients without PPGL. Patients with falsely elevated baseline NMN results (n = 7, 26.9%) exhibited increases of baseline NMN up to 1.7-fold above the upper reference limit. CONCLUSION: CST qualifies as a useful diagnostic tool for differential diagnosis of borderline elevated plasma-free NMN in patients with suspected PPGL. In this context, CST helps to correctly identify all false-positive NMN screening results.

Head/neck paragangliomas: focus on tumor location, mutational status and plasma methoxytyramine.

Head and neck paragangliomas (HNPGLs) are tumors of parasympathetic origin that occur at variable locations and are often secondary to germline mutations in succinate dehydrogenase (SDH) subunit genes. Occasionally, these tumors produce catecholamines. Here, we assessed whether different locations of HNPGLs relate to the presence of SDHx mutations, catecholamine production and other presentations. In this multicenter study, we collected clinical and biochemical data from 244 patients with HNPGLs and 71 patients without HNPGLs. We clarified that jugulotympanic HNPGLs have distinct features. In particular, 88% of jugulotympanic HNPGLs arose in women, among whom only 24% occurred due to SDHx mutations compared to 55% in men. Jugulotympanic HNPGLs were also rarely bilateral, were of a smaller size and were less often metastatic compared to carotid body and vagal HNPGLs. Furthermore, we showed that plasma concentrations of methoxytyramine (MTY) were higher (P < 0.0001) in patients with HNPGL than without HNPGL, whereas plasma normetanephrine did not differ. Only 3.7% of patients showed strong increases in plasma normetanephrine. Plasma MTY was positively related to tumor size but did not relate to the presence of SDHx mutations or tumor location. Our findings confirm that increases in plasma MTY represent the main catecholamine-related biochemical feature of patients with HNPGLs. We expect that more sensitive analytical methods will make biochemical testing of HNPGLs more practical in the future and enable more than the current 30% of patients to be identified with dopamine-producing HNPGLs. The sex-dependent differences in the development of HNPGLs may have relevance to the diagnosis, management and outcomes of these tumors.

Quantification of normetanephrine in canine urine using ELISA: evaluation of factors affecting results.

Catecholamine release increases in dogs with pheochromocytomas and in situations of stress. Although plasma catecholamines degrade rapidly, their metabolites, normetanephrine (NME) and metanephrine (ME), are stable in acidified urine. Our aim was to verify a human urine ELISA kit for the quantification of NME and ME in canine urine and to determine the effects on metabolite stability of sampling time (morning or midday) and day (ordinary or day spent in a clinic). We analyzed 179 urine samples from 17 healthy dogs. For NME, the mean intra-assay CV was 6.0% for all samples and 4.3% for the canine control; inter-assay CVs were 3.3, 3.8, and 12% for high and low concentration human urine positive controls supplied in the ELISA kit and a positive canine control, respectively; spike-recovery was 90-101%. For ME, mean intra-assay CV was 6.5% for samples and 9.0% for the canine control; inter-assay CVs were 12.7, 7.2, and 22.5% for high and low concentration human urine positive controls supplied in the ELISA kit and a positive canine control, respectively; spike-recovery was 85-89%. Dilution recovery was unsatisfactory for both metabolites. Based on our verification results, NME was selected for remaining analyses. We found no effect on NME concentrations of acidification or room temperature storage for up to 24 h. The NME:creatinine ratio was higher after the first of 3 clinic days compared to the same morning (111.2 ± 5.5 vs. 82.9 ± 5.3; p < 0.0001), but not on the other days. NME verification results were generally superior to ME. Dilution studies were unsatisfactory for both metabolites. Given that NME was stable without acidification at room temperature, urine samples can be collected at home. The clinic environment can cause higher NME:creatinine ratios, especially in unaccustomed dogs.

Updated reference intervals for urine normetanephrine have no effect on test sensitivity but fewer false positives.

OBJECTIVE: We previously highlighted the problem of frequent false positives in 24 h urine normetanephrine(UNM) measurements owing to reference intervals that are inappropriately low for the population being screened for pheochromocytoma. Using a large population database, we devised new age-stratified reference intervals for the 24 h UNM test that were higher compared to previous. However, it was uncertain as to whether this would compromise test sensitivity for true pheochromocytoma cases. DESIGN AND METHODS: Retrospective analysis of all pheochromocytoma cases from a recently constructed provincial registry. All confirmed cases had their diagnostic UNM results retrospectively re-analysed according to the newly proposed UNM reference intervals to determine the percentage and phenotype of cases that might have been theoretically missed with the new reference range. RESULTS: After excluding pediatric and non-secretory head and neck paragangliomas, there were 60 confirmed pheochromocytoma cases. Using prior reference intervals, 51/60 (85%) had an abnormally high UNM. Of the 9 with normal UNM, 4 had a high urine metanephrine(UMN), 5 had normal levels of both UNM and UMN such that 55/60 had abnormal test results, representing the historical combined test sensitivity of 92%. Using the proposed reference interval, 43/60 (72%) had high UNM results. Of the 17 with normal UNM, 12 had high UMN, 5 had normal levels of both UNM and UMN. Therefore, 55/60 patients had had elevations in either UNM or UMN, corresponding to an identical combined test sensitivity of 92%. CONCLUSIONS: Reference intervals for UNM derived from actual clinical population screening data are higher than in traditional healthy volunteers. Use of these more appropriate reference intervals can significantly reduce the false positive rate without compromising test sensitivity for true pheochromocytoma.

A high sensitivity LC-MS/MS method for measurement of 3-methoxytyramine in plasma and associations between 3-methoxytyramine, metanephrines, and dopamine.

INTRODUCTION: Diagnosis of pheochromocytoma and paraganglioma (PPGL) is aided by the measurement of metanephrine (MN) and normetanephrine (NMN). Research suggests that 3-methoxytyramine (3MT), a dopamine (DA) metabolite, may serve as a biomarker of metastasis in patients with paraganglioma. Considering the very low endogenous plasma 3MT concentrations (<0.1 nM), highly sensitive and specific methods for 3MT are needed. METHODS: We developed a simple method for measurement of 3MT. Sample preparation was performed using solid phase micro-extraction with the eluates injected directly onto the LC-MS/MS. Data acquisition was performed in multiple reaction monitoring mode with an instrumental analysis time of 3 min per sample. We evaluated the method's performance and analyzed samples from healthy individuals and pathological specimens. RESULTS: The limit of quantitation and upper limit of linearity were 0.03 nM and 20 nM, respectively. The intra-/inter-day imprecision for pooled plasma samples at concentrations of 0.04 nM, 0.2 nM, and 2 nM was 10.7%/18.3%, 4.5%/8.9%, and 3.1%/0.9%, respectively. Among samples with MN, NMN, or both MN and NMN above the reference intervals (RIs), 0%, 16% and 46%, respectively, showed 3MT greater than the proposed upper RI value of 0.1 nM; 12% of samples with DA above the RI had 3MT above 0.1 nM. CONCLUSIONS: The developed method allowed accurate quantitation of 3MT in patient samples and would provide valuable information to clinicians diagnosing or monitoring patients with PPGL. High 3MT concentrations in patient samples with MN and NMN within the respective RIs may alert clinicians of the possibility of a DA-producing tumor.

Study of stability and interference for catecholamines and metanephrines, 3-methoxytyramine: key point of an accurate diagnosis for pheochromocytoma and paraganglioma.

BACKGROUND: Accurate diagnosis of pheochromocytoma and paraganglioma (PPGLs) is highly dependent on the detection of metanephrines and catecholamines. However, the systematic investigation on influencing factors including specimen (plasma or whole blood), anticoagulant, storage conditions, and interference factors need further confirmation. METHODS: Blood with heparin-lithium or EDTA-K2 were collected, stability of epinephrine (EPI), norepinephrine (NE), dopamine (DA), metanephrine (MN), normetanephrine (NMN), 3-methoxytyramine (3-MT) in whole blood and plasma at room temperature and 4 °C for different storage times, stability of plasma MN, NMN and 3-MT at -20 °C and -80 °C were investigated. Plasma with hemoglobin (1 g/L, 2 g/L, 3 g/L, 4 g/L, 6 g/L), TG (<5 mmol/L, 5-8 mmol/L, >8 mmol/L) were prepared. RESULTS: EPI, NE, DA were prone to degrade at room temperature, samples should be centrifuged at 4 °C. EPI and NE were stable in whole blood at 4 °C for 4 h and in plasma for 2 h. For MN, NMN, 3-MT, plasma can be stable at room temperature and 4 °C for at least 6 h, which is better than whole blood; there was no significant difference when stored at -20 °C and -80 °C for 7 days. Heparin-lithium had a slight advantage over EDTA-K2. EPI, NE, DA should not be performed when Hb > 1 g/L or TG > 5 mmol/L. MN, NMN, 3-MT should not be performed when Hb > 2 g/L, whereas TG had no interference. CONCLUSIONS: According to the actual clinical application scenario, this study provided a reliable basis for the accurate diagnosis of PPGLs.