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The urinary normetanephrine to creatinine ratio (uNMN/Cr) and urinary metanephrine to creatinine ratio (uMN/Cr) are commonly evaluated for the diagnosis of pheochromocytomas (PCC) in dogs. This study aimed to establish reference intervals for uNMN/Cr and uMN/Cr in 56 healthy dogs in Japan and to investigate the effect of urine collection methods on these measurements in 15 non-PCC dogs. The upper limits of reference intervals for uNMN/Cr and uMN/Cr were 124.4 nmol/mmol (90% confidence interval [CI] 107.7-137.0) and 121.1 nmol/mmol (90% CI 102.8-145.1), respectively. Both uNMN/Cr and uMN/Cr were significantly higher when urine was collected in the hospital compared to at home. Several factors, including the method of urine collection, should be considered when utilizing these reference intervals.

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., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Urine metanephrines are used to screen for phaeochromocytoma or paraganglioma (PPGL). Current reference intervals (RI) derived in healthy individuals are not age or sex-stratified, and lower than in hypertensive patients, leading to high false positive rates. This study aims to determine age and sex-stratified RI from a contingent screening population., Methods: Patients with 24-h deconjugated urine metanephrines from 3/6/2010 to 27/8/2022 were included (2936 males, 5285 females), initially by liquid chromatography-electrochemical detection (LC-ECD) then liquid chromatography-tandem mass spectrometry (LC-MS/MS). Bhattacharya analysis was used after log transformation to determine age and sex-stratified RI for metanephrine excretion, normetanephrine excretion, metanephrine/creatinine and normetanephrine/creatinine ratios., Results: Normetanephrine excretion increases with age (RI: males: 18-<30 years: <3.4 µmol/24 h, 30-<40 years: <3.7 µmol/24 h, 40+ years: <5.3 µmol/24 h; females: 18-<30 years: <2.7 µmol/24 h, 30-<40 years: <3.1 µmol/24 h, 40+ years: <3.7 µmol/24 h), while metanephrine excretion was consistent across adulthood (RI: males: 18+ years: <1.8 µmol/24 h; females: 18+ years: <1.2 µmol/24 h). However, normetanephrine/creatinine and metanephrine/creatinine increase steadily with age after early adulthood, likely due to a decrease in muscle mass, with females having higher normetanephrine/creatinine and metanephrine/creatinine ratios., Conclusions: Age and sex-stratified RI were derived for metanephrine excretion, normetanephrine excretion, metanephrine/creatinine and normetanephrine/creatinine ratios. This is expected to reduce false positives while flagging most PPGL., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Pheochromocytoma (PHEO) is a tumor from the chromaffin tissue of the adrenal medulla, capable of hyperproduction of catecholamines. The increased production of hormones by the tumor leads to catecholamine crises, which have a pathological effect on all organs and systems. In the primary diagnosis of pheochromocytomas, it is important to determine the level of the metabolite of catecholamines - metanephrines. Currently, in clinical practice, various methods are used to determine the level of this metabolite: in blood plasma or in urine, total or only free form, fractionated analysis or unfractionated., Aim: Comparison of the effectiveness of various methods for determining the level of metanephrines for the diagnosis of pheochromocytomas., Materials and Methods: A retrospective single-center cohort study was conducted on a sample of patients who were initially operated on for adrenal neoplasm at the Pirogov St. Petersburg State University High Medical Technology Clinic from November 2007 to December 2022 and who passed analysis to determine the level of blood or urine metanephrins before surgical treatment. The results of tests for metanephrine and tumor size were evaluated., Results: 1088 patients with adrenal neoplasms who underwent surgical treatment were examined, of which 348 had histologically confirmed the presence of pheochromocytoma. Four types of metanephrine assays were compared: free fractionated plasma metanephrines (232 patients), unfractionated daily urine metanephrines (431 patients), fractionated total daily urine metanephrines (427 patients) and fractionated free daily urine metanephrines (178 patients). The greatest sensitivity was demonstrated by the analysis of free fractionated plasma methanephrines (95.4%). Unlike others, the sensitivity of this analysis did not decrease in the group of patients with small pheochromocytomas (3 cm or less). The greatest specificity was demonstrated by the analysis of unfractionated metanephrines in daily urine (97.8%), with the lowest sensitivity among all tests (67.6%). The study of fractionated total daily urine metanephrins showed good results of sensitivity and specificity, only slightly inferior to the best indicators, and the analysis of free daily urine metanephrins demonstrated unexpectedly low efficiency. There is a positive correlation between the level of metanephrine in the blood and the size of the tumor., Conclusion: Based on the data obtained, the preferred assays for the primary diagnosis of pheochromocytoma can be considered the determination of fractionated free plasma metanephrines and fractionated total daily urine metanephrines, which is consistent with relevant clinical recommendations. It was found that the size of the tumor correlates with the severity of an increase in the level of metanephrins determined by any of the described methods.

Background: Sparse metanephrines reference intervals in pediatric populations are available and different study designs and technologies/ assays used in these studies lead to hardly transferable data from a laboratory to another. The aim of this study was to update pediatric reference intervals of total fractionated metanephrines in spot urine samples, using a commercial extraction kit run on a specific high-pressure liquid chromatograph coupled with an electrochemical detector., Methods: Four hundred and fifty-two spot pediatric urinary samples previously submitted to urinalysis were consecutively included in the study with the exclusion of children's samples with diagnosis or clinical suspicion of paraganglioma/pheochromocytoma, kidney diseases and arterial hypertension. Urinary metanephrine, normetanephrine and 3-methoxytyramine were extracted with ClinRep ® HPLC Complete kit and run on HPLC Prominence liquid chromatograph LC-20AT (Shimadzu Italia S.r.l. Milan, Italy) coupled with Decade II electrochemical detector (Antec Scientific, Zoeterwoude, the Nederlands, provided by Alfatech S.r.l., Genoa, Italy). Results were expressed as the ratio analyte-to-creatinine., Results: Any of the three analytes required a repartition by gender (metanephrine P=0.27; normetanephrine P=0.90 and 3-methoxytyramine P=0.18). A significant statistically inversely proportional relation with age was found for metanephrine (P<0.0001; ρ=-0.72), normetanephrine (P<0.0001; ρ=-0.75) and 3-methoxytyramine (P<0.0001; ρ=-0.83). Reference intervals were calculated as function of age., Conclusions: This study provides pediatric reference intervals for urinary fractionated total metanephrines in spot urine calibrated on a specific instrumentation and extraction commercial kit.

Objectives: Biochemical testing of urinary metanephrines is useful in the diagnosis and monitoring of pheochromocytoma and paragangliomas. We investigated the feasibility of mixture decomposition (ie, indirect) methods in verifying clinically derived reference intervals for urinary deconjugated metanephrine metabolites., Methods: Urinary 24-hour metanephrine and normetanephrine excretion results were extracted from our data warehouse and intervals were estimated by the modern variant of the Hoffmann method, maximum likelihood estimation (MLE), and gamma mixture model using R software., Results: Hoffmann, MLE, and gamma mixture models provided metanephrine and normetanephrine intervals that closely matched those derived from clinical studies. However, three-component MLE and gamma models were required for normetanephrine in adult women because the Hoffmann method was not suitable. Some data transformations caused blending of the mixed distributions and subsequent widening of the reference interval estimation, emphasizing the importance of careful data transformation for Hoffmann and MLE analyses. Gamma mixture models gave overall good agreement without the need for data transformation., Conclusions: Indirect methods have utility in verifying reference intervals in 24-hour urine specimens collected by patients. We emphasize the benefits of applying multiple decomposition methods to corroborate findings and careful application of data transformation when using Gaussian-based models., (© American Society for Clinical Pathology, 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Pheochromocytoma is a rare cause of acute cardiovascular disease; however, any severe illness may have high catecholamines, simulating pheochromocytoma. We determined the spectrum of urine metanephrines from inpatient and outpatient collections without pheochromocytoma, compared with confirmed pheochromocytoma patients., Methods: Retrospective analysis using centralized laboratory data serving all outpatients and hospitals in southern Alberta. The analysis comprised 24-hour urine normetanephrine and metanephrine (UNM-UMN) results collected from hospital inpatients, community outpatients, and patients from a comprehensive provincial pheochromocytoma registry., Results: There were 974 unique inpatients (including 132 from intensive care), 6802 outpatients, and 58 pheochromocytoma patients. Among outpatient, general ward, and intensive care unit (ICU) patients, 18.7%, 34.4%, and 67.4% of results, respectively, were supranormal. Although pheochromocytoma patients had higher median UNM-UMN vs inpatients, there was substantial overlap. Receiver operating characteristic (ROC) analysis showed area under the curve (AUC) of 0.64-0.91 to detect true pheochromocytoma (P < .0001), with progressively poorer discrimination among hospitalized and ICU-dependent patients. A 24-hour urine normetanephrine >6.95 nmol/d had 98% specificity for pheochromocytoma when inpatient general ward samples were included, but only 46% sensitivity and 13% positive predictive value for pheochromocytoma. Considering ICU collections, 98% specificity required results more than fivefold above the upper reference limit and still had poor positive predictive value. A model combining both UNM and UMN results as a cross-product marginally improved the ROC AUC, but improved sensitivity in outpatients and ward patients but not ICU patients., Conclusion: There is a high degree of overlap in UNM-UMN between hospitalized patients and pheochromocytoma; high test specificity is not achieved in this population unless >3-5 times the upper reference limit., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Context: False-positive results are common for pheochromocytoma/paraganglioma (PPGL) real-world screening., Objective: Determine the correlation between screening urine and seated plasma metanephrines in outpatients where PPGL was absent, compared to meticulously prepared and supine-collected plasma metanephrines with age-adjusted references., Design: Retrospective cohort study., Setting: Databases from a single-provider provincial laboratory (2012-2018), a validated PPGL registry, and a manual chart review from a specialized endocrine testing unit., Patients: PPGL registry data excluded known PPGL cases from the laboratory database. Outpatients having both urine and plasma metanephrines <90 days apart., Methods: The correlation between urine and seated plasma measures along with the total positivity rate. All cases of plasma metanephrines drawn in the endocrine unit were reviewed for test indication and test positivity rate., Results: There were 810 non-PPGL pairs of urine and plasma metanephrines in the laboratory database; 46.1% of urine metanephrines were reported high. Of seated outpatient plasma metanephrines drawn a median of 5.9 days later, 19.2% were also high (r = 0.33 and 0.50 for normetanephrine and metanephrine, respectively). In contrast, the meticulously prepared and supine collected patients (n = 139, 51% prior high urine metanephrines) had <3% rate of abnormal high results in patients without known PPGL/adrenal mass., Conclusions: There was a poor-to-moderate correlation between urine and seated plasma metanephrines. Up to 20% of those with high urine measures also had high seated plasma metanephrines in the absence of PPGL. Properly prepared and collected supine plasma metanephrines had a false-positive rate of <3% in the absence of known PPGL/adrenal mass., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Catecholamines are physiological regulators of carbohydrate and lipid metabolism during stress, but their chronic influence on metabolic changes in obese patients is still not clarified. The present study aimed to establish the associations between the catecholamine metabolites and metabolic syndrome (MS) components in obese women as well as to reveal the possible hidden subgroups of patients through hierarchical cluster analysis and principal component analysis. The 24-h urine excretion of metanephrine and normetanephrine was investigated in 150 obese women (54 non diabetic without MS, 70 non-diabetic with MS and 26 with type 2 diabetes). The interrelations between carbohydrate disturbances, metabolic syndrome components and stress response hormones were studied. Exploratory data analysis was used to determine different patterns of similarities among the patients. Normetanephrine concentrations were significantly increased in postmenopausal patients and in women with morbid obesity, type 2 diabetes, and hypertension but not with prediabetes. Both metanephrine and normetanephrine levels were positively associated with glucose concentrations one hour after glucose load irrespectively of the insulin levels. The exploratory data analysis showed different risk subgroups among the investigated obese women. The development of predictive tools that include not only traditional metabolic risk factors, but also markers of stress response systems might help for specific risk estimation in obesity patients.

Urinary catecholamines and their methylated metabolites are biochemical indicators of pheochromocytoma, paraganglioma and neuroblastoma. A rapid and precise analytical method based on solid-phase extraction (SPE) and liquid chromatography separation coupled to high-resolution mass spectrometry (LC-HRMS) was developed and validated to measure urinary catecholamines (epinephrine (E), norepinephrine (NorE), dopamine (D)) and total methylated metabolites (normetanephrine (NorMN), metanephrine(MN) and 3-methoxytyramine (3-MT)) in a clinical setting. Results of 51 urine specimens measured using this LC-HRMS method were compared with a liquid chromatography assay with electrochemical detection (LC-EC). Urine samples (200 μL) were spiked with an internal standard solution followed by SPE purification. In the case of total methylated metabolites, urine was hydrolyzed before SPE purification. Separation was achieved on an Acclaim Mixed Mode WCX column, with an 8.5 min runtime. All compounds were detected in electrospray positive ionization mode with a parallel reaction monitoring acquisition and quantified with a linear regression (r2 > 0.998) between 2 and 200 µg/L (10.9-1090; 11.8-1182 nmol/L) for E and NorE respectively and between 10 and 1000 µg/L for others (65.2-6520; 50.7-5070; 54.5-5450 ; 59.8-5980 nmol/L for D, M, NorMN and 3-MT, respectively). Overall imprecision and bias did not exceed 15%. No significant matrix effect was observed. Correlation between the two assays was good except for epinephrine. Epinephrine concentrations measured by LC-EC method were slightly higher than values obtained with LC-HRMS method but without impact on clinical decision. This LC-HRMS assay provides a new tool for simultaneous quantitative catecholamine determination and was successfully applied in routine for the screening or follow up of pheochromocytoma, paraganglioma and neuroblastoma. LC-HRMS method offers significant advantages compared to LC-EC with good sensitivity, an unambiguous analyte determination and high sample throughput., (Copyright © 2020 Elsevier B.V. All rights reserved.)

One pitfall in 24-hour urine collection is the input of incorrect urinary volume by the reference laboratory. This may lead to an incorrect diagnosis of pheochromocytoma or paraganglioma. A 48-year-old African-American woman was seen in the clinic for an elevated 24-hour urine metanephrine screen during workup for secondary hypertension. Urine volume was found to be incorrectly inputted by the lab as 9750 mL rather than 975 mL. The urinary metanephrines were then recalculated and the 24-hour urinary metanephrines resulted within normal limits. This case highlights this unique and potentially under-recognised error in testing with 24-hour urine volume collection., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Detection of normetanephrine (NMN), metanephrine (MN) and 3-methoxytyramine (3-MT) could be used to diagnose pheochromocytomas and paragangliomas (PPGLs). The accuracy for the diagnosis of PPGLs is only 6% by virtue of the classic symptom triad. In addition, false-positive results were found using plasma free MNs as biomarkers. Spot urinary free metanephrines (MNs) presented high specificity for PPGLs diagnosis in our previous work by HPLC with the electrochemical detection. Whereas, MNs and creatinine (Cr) need to be detected separately. A simple and specific method was urgently needed for the diagnosis of PPGLs. Here, we established a new HPLC method for spot urinary free MNs and 3-MT by the fluorescence detection and Cr by the ultraviolet detection simultaneously. It was worth mentioning that Cr for the virtue of being fairly constant in a given subject was used as an internal reference correction to eliminate the effect of spot urine volume for the diagnosis of PPGLs. Thirty-seven patients with PPGLs and 164 control subjects were detected by the established method and the peak area ratios of MNs and 3-MT to Cr were used innovatively for the diagnosis of PPGLs. The results showed acceptable precisions and recoveries. The sensitivities of the method were 94.6%, 91.9% and 86.5% and the specificities were 96.3%, 93.9% and 82.3%, respectively by the peak area of NMN/Cr, MN/Cr and 3-MT/Cr for the diagnosis. The established method provides a promising way for simple, rapid and accurate diagnosis of PPGLs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: In patients with phaeochromocytomas or paragangliomas (PPGLs), 24-h urine collections for metanephrines (uMNs) are cumbersome., Objective: To evaluate the diagnostic utility of ratios to creatinine of 'spot' uMNs., Methods: Concentrations of uMNs and plasma metanephrines (pMNs) were measured by HPLC-mass-spectrometry. We retrospectively compared correlations of 24-h-urine output and ratio to creatinine in historical specimens and prospectively assessed 24-h and contemporaneous spot urines and, where possible, pMNs. Using trimmed log-transformed values, we derived reference intervals based on age and sex for spot urines. We used multiples of upper limit of normal (ULNs) to compare areas under curves (AUCs) for receiver-operator characteristic curves of individual, and sum and product of, components., Results: In 3143 24-h-urine specimens on 2416 patients, the correlation coefficients between the ratios and outputs of metanephrine, normetanephrine and 3-methoxytyramine in 24-h urines were 0.983, 0.905 and 0.875, respectively. In 96 patients, the correlations between plasma concentrations, urine output and ratios in spot specimens were similar to those for raw output or ratios in 24-h specimens. Of the 160 patients with PPGLs, the CIs for AUCs for individual metabolites overlapped for all four types of measurement, as did those for the sum of the multiple ULNs although these were slightly higher (AUC for spot urine: 0.838 (0.529-1), plasma: 0.929 (0.874-0.984) and output: 0.858 (0.764-0.952))., Conclusions: Ratios of fractionated metanephrines to creatinine in spot urine samples appear to have a similar diagnostic power to other measurements. The ease of spot urine collection may facilitate diagnosis and follow-up of PPGLs through improved patient compliance.

A 63-year-old woman with multiple endocrine neoplasia type 2A (MEN2A) presented with recurrent spells of headaches, sweats and palpitations decades after right adrenalectomy for phaeochromocytoma, and total thyroidectomy for medullary thyroid cancer. She was hypertensive and in sinus rhythm. DOTA-TATE positron-emission tomography (PET) demonstrated a 12mm enhancing left adrenal incidentaloma. 24 hours urine catecholamines, and multiple plasma metanephrine and normetanephrine measurements were all within normal reference ranges. Based on her symptoms and imaging findings, left adrenalectomy was performed and found a 40 mm phaeochromocytoma. Her symptoms have since completely resolved and plasma metanephrine is now undetectable MEN2-associated phaeochromocytomas are often bilateral and may be metachronous. Patients at high risk of phaeochromocytoma who develop symptoms of catecholamine excess should be carefully evaluated even if plasma or urinary metanephrines are within the normal reference range. Biochemical reference ranges for metanephrines need to be adjusted accordingly in patients who have had prior unilateral total adrenalectomy., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Urinary measurement of fractionated metanephrines is one of the best available biochemical tests for the diagnosis of pheochromocytoma and paraganglioma; however, false-positive results remain an issue. In addition, despite their convenience, there is no evidence to suggest that spot urine samples should replace the standard 24-h urine collection method. The objective of this study is to elucidate clinical variables that may influence the levels of spot urine fractionated metanephrines and to examine their instability. The study is a retrospective review of prospectively collected data from 949 patients whose spot urine fractionated metanephrines were measured under suspicion of pheochromocytoma or paraganglioma at our institution from January 2011 to June 2017. The effects of clinical factors such as age, sex, renal function, and medications on the level of spot urine fractionated metanephrines were evaluated. Urinary fractionated metanephrines were significantly higher in female subjects. They correlated with age but not with estimated glomerular filtration rate (eGFR). A multivariate regression model for urinary fractionated metanephrine levels revealed that age, female sex, eGFR, and the presence of α1-blocker medication were significantly positively correlated with urinary metanephrine (M) levels. Age, female sex, eGFR, and presence of α1-blockers and antidepressant and antipsychotic medications positively correlated with urinary normetanephrine (NM). In addition, age, female sex, eGFR, and presence of α1-blockers and antidepressant and antipsychotic medications were positively correlated with urinary M + NM. In conclusion, sex, age, eGFR, and treatment with α1-blockers, antidepressants, and antipsychotics may affect the levels of urinary fractionated metanephrines. When assessing urinary fractionated metanephrines in patients with suspected PPGLs, we must consider these factors.

Pheochromocytoma and paraganglioma (PPGL) are rare neuroendocrine tumors arising from adrenal and extra-adrenal chromaffin cells. They produce excessive amounts of catecholamines and their metabolites. A newly analytical procedure based on the semi-automated microextraction by packed sorbent (MEPS) technique, using a digitally controlled syringe (eVol) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), was developed to quantify free urinary catecholamines and metanephrines. The important parameters affecting MEPS performance, namely the type of sorbent material (porous graphitized carbon (PGC), polar enhanced polymer (PEP), cation-exchange (CX) and C18), number of extraction cycles, and elution solvent system, were evaluated. The optimal experimental conditions involved the loading of sample mixture in seven extraction cycles through a C18 sorbent in a MEPS syringe, followed by using elution solutions (water/acetonitrile/formic acid, 95/4.75/0.25). The entire sample preparation took about 4 min. Chromatographic separation was well achieved with an HSS PFP column using the gradient elution. The linearity range of the method was 0.167-33.4 ng/mL for epinephrine, 0.650-130 ng/mL for norepinephrine, 1.53-306 ng/mL for dopamine, 1.34-268 ng/mL for metanephrine, 3.43-686 ng/mL for normetanephrine, and 1.33-265 ng/mL for 3-methoxytyramine. The intra- and interassay precisions were ≤ 12.8%, and the respective accuracies were 88.4-112.0% and 89.0-109.5%. The carryover and sample stability without acidification were also investigated. Validation using clinical urine specimens showed that the proposed method had higher sensitivity compared with other urinary biochemical tests. The developed MEPS-LC-MS/MS method was simple, fast, and cost-effective; it helped to obtain information about multiple metabolites. It is applicable in routine clinical laboratories for the screening of PPGL. Graphical abstract.

Objective: Sympathoadrenal activity is decreased during overnight rest. This study assessed whether urinary-free normetanephrine, metanephrine and methoxytyramine in overnight/first-morning urine collections might offer an alternative to measurements in 24-h collections or plasma for diagnosis of pheochromocytoma and paraganglioma (PPGL)., Design and Methods: Prospective multicenter cross-sectional diagnostic study involving 706 patients tested for PPGL, in whom tumors were confirmed in 79 and excluded in 627 after follow-up. Another 335 age- and sex-matched volunteers were included for reference purposes. Catecholamines and their free O-methylated metabolites were measured in 24-h collections divided according to waking and sleeping hours and normalized to creatinine. Plasma metabolites from blood sampled after supine rest were measured for comparison., Results: Urinary outputs of norepinephrine, normetanephrine, epinephrine and metanephrine in the reference population were respectively 50 (48-52)%, 35 (32-37)%, 76 (74-78)% and 15 (12-17)% lower following overnight than daytime collections. Patients in whom PPGLs were excluded showed 28 (26-30)% and 6 (3-9)% day-to-night falls in normetanephrine and metanephrine, while patients with PPGLs showed no significant day-to-night falls in metabolites. Urinary methoxytyramine was consistently unchanged from day to night. According to receiver-operating characteristic curves, diagnostic accuracy of metabolite measurements in overnight/first-morning urine samples did not differ from measurements in 24-h urine collections, but was lower for both than for plasma. Using optimized reference intervals, diagnostic specificity was higher for overnight than daytime collections at similar sensitivities., Conclusions: Measurements of urinary-free catecholamine metabolites in first-morning/overnight urine collections offer an alternative for diagnosis of PPGL to 24-h collections but remain less accurate than plasma measurements.

Background: Urinary metanephrine is a reliable method to estimate catecholamine secretion. Traditionally, urinary metanephrines are collected into chilled containers containing hydrochloric acid (HCl) and most laboratories freeze urinary samples before analysis. It is uncertain if these pre-analytic procedures alter metanephrine values., Aim: To evaluate if acidifying and freezing urine samples affect the accuracy of urinary metanephrine measurements., Methods: Random urine samples from healthy individuals were collected. Urine samples were distributed into two containers: with HCl 50% homogenized with urine to obtain pH < 2, and without HCl. Each container was divided again into aliquots for immediate measurement or freezing. One aliquot with acid (group 1) and another without acid (group 2) were sent immediately to the laboratory for testing (HPLC), while the other two aliquots, one with acid (group 3) and another without it (group 4) were frozen for 3 months at - 20 °C. Bland-Altman's test was used to analyze inter-assay agreement between measurements., Results: A total of 15 individuals were included (mean age 27.5 ± 5.9 years, 8 male and 14 white). No difference was observed on mean urinary metanephrine/creatinine ratio between groups: group 1: 0.23 ± 0.11, group 2: 0.22 ± 0.07, group 3: 0.25 ± 0.13, group 4: 0.25 ± 0.15 mg/g creatinine; P > 0.05 for all the comparisons). Bland-Altman's analysis showed agreement between the standard method (group 1) and the experimental method (group 4)., Conclusion: Measurement of urinary metanephrines by HPLC method is not influenced by sample acidification nor freezing at - 20 °C for 3 months.

We recently conducted an open-label phase I/II study to evaluate the efficacy and safety of preoperative and chronic treatment with metyrosine (an inhibitor of catecholamine synthesis) in pheochromocytoma/paraganglioma (PPGL) in Japan. We compared creatinine-corrected metanephrine fractions in spot urine and 24-hour urine samples (the current standard for the screening and diagnosis of PPGLs) from 16 patients to assess the therapeutic effect of metyrosine. Percent changes from baseline in urinary metanephrine (uMN) or normetanephrine (uNMN) were compared between spot and 24-hour urine samples. Mean percent changes in uMN or uNMN in spot and 24-hour urine were -26.36% and -29.27%, respectively. The difference in the percent change from baseline between uMN or uNMN in spot and 24-hour urine was small (-2.90%). The correlation coefficient was 0.87 for percent changes from baseline between uMN or uNMN measured in spot and 24-hour urine. The area under the receiver operator characteristic (ROC) curve of uMN or uNMN measured in spot urine vs. 24-hour urine (reference standard) to assess the efficacy of metyrosine treatment was 0.93. Correlations and ROCs between 24-hour urinary vanillylmandelic acid, adrenaline, and noradrenaline and 24-hour uMN or uNMN were similar to those between spot uMN or uNMN and 24-hour uMN or uNMN. No large difference was observed between spot and 24-hour urine for the assessment of metyrosine treatment by quantifying uMN or uNMN in Japanese patients with PPGLs. These results suggest that spot urine samples may be useful in assessing the therapeutic effect of metyrosine.

The diagnostic utility of different thresholds of elevated urinary fractionated metanephrine (UFM) for pheochromocytoma-paraganglioma (PPGL) was evaluated in 10 164 community-dwelling subjects (2012-2017). Levels were ≥1.5× the upper normal limit (UNL) in 276 subjects (2.7%) and ≥2×UNL in 138 (1.4%). PPGL was subsequently diagnosed in 59 (mean age 51.9 ± 14.3, 64% female); 58 (98.3%) with UFM ≥ 2×UNL. Positive predictive values (PPV) were 42% for UFM ≥ 2×UNL, 55% for UFM ≥ 2.5×UNL, and 69% for UFM ≥ 3×UNL. The main reason for PPGL screening (52.5%) was adrenal incidentaloma. Mean (median) metanephrine/normetanephrine levels were 6.7 ± 9×UNL (3 × UNL) and 6.1 ± 8.9×UNL (2.5 × UNL). Six patients (10.2%) had an extra-adrenal tumor (one malignant paraganglioma); one had bilateral pheochromocytoma. Only one patient presented with the "classic triad" (headache, palpitations, sweating). In conclusion, after excluding obvious reasons for false-positive results, thorough diagnostic assessment for PPGL is justified in all subjects with UFM ≥ ×2UNL. The PPV of milder UFM elevations is very low., (©2019 Wiley Periodicals, Inc.)

Compared with the general population, rates of pheochromocytoma are higher in neurofibromatosis type 1 (NF1) patients. However, pheochromocytoma testing is often plagued by false positive results. Here we present a patient with NF1, elevated urinary metanephrine levels, and an indeterminate adrenal nodule. Clonidine suppression testing aided diagnosis and led to definitive surgical treatment that confirmed a pheochromocytoma. Pheochromocytoma screening and clonidine suppression testing can both aid in the evaluation for catecholamine-secreting tumours., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: Pheochromocytoma and paraganglioma are uncommon tumors whose best known symptoms include high blood pressure, palpitations, headache, and sweating. Clinical identification is not easy, however, and requires biochemical tests that allow for early diagnosis, including measurement of metanephrines levels. The aim of this study was to assess the diagnostic performance of plasma free metanephrines (PMETs) and to verify the transferability of the reference values used., Methods: PMETs levels were measured by liquid chromatography coupled to tandem mass spectrometry. Other biochemical tests evaluated (plasma catecholamine, urine metanephrine, catecholamine and vanilmandelic acid levels) were performed by liquid chromatography with electrochemical detection. Requests of these tests from 01/09/2015 to 31/10/2017 were reviewed, and both the reference values (document EP28-A3c) and the parameters of biological variation (Fraser method) for PMETs were estimated., Results: The study sample consisted of 1,279 patients (61.3% females) aged 0-90 years, including 19 with pheochromocytoma/paraganglioma. Tests requested included: PMETs (n=662), catecholamines (n=589), metanephrines (n=586), and vanilmandelic acid (n=513) in urine, and plasma catecholamines (n=228). Tests with higher sensitivity were urinary fractionated metanephrines (91.7%) and PMETs (82.4%). When performance was compared in patients with both tests (n=243), they detected the same number of tumors (90.9%), but PMETs showed greater specificity (93.5% vs 88.8%). Plasma normetanephrine levels showed a significant association with age (rho=0.19, P<.0001)., Conclusion: PMETs and urinary fractionated metanephrines are the biochemical tests with better performance in diagnosis of pheochromocytomas/paragangliomas., (Copyright © 2018 SEEN y SED. Publicado por Elsevier España, S.L.U. All rights reserved.)

Introduction: A substantial number of patients with neuroblastoma (NB) have increased excretion of catecholamines and metanephrines. Here, we have investigated the diagnostic role of plasma free metanephrines (PFM), metanephrine (MN), normetanephrine (NMN) and 3-methoxytyramine (3MT) for NB, the most common extra-cranial solid tumour in children., Methods: PFM were quantified by using a commercial IVD-CE LC-MS/MS method on a TSQ Quantiva coupled to an Ultimate 3000. The method was further validated on 103 samples from pediatric subjects (54 patients with histologically confirmed NB and 49 age and sex matched controls). Correlations between PFM concentrations with clinical factors were tested. We directly compared MN, NMN, and 3MT concentrations in matched plasma and urine samples of NB patients (n = 29)., Results: 3MT and NMN showed an excellent diagnostic performance with very high specificity (100% and 95.8%, respectively) and sensitivity (88.2% and 80.4%). ROC curves were obtained (AUC of 0.93 and 0.91 for 3MT and NMN, respectively) and optimal cut-offs that could discriminate between controls and NB patients were defined. A positive correlation between NMN levels in urine and plasma (p = .0017) was found., Discussion: The determination of plasma 3MT and NMN should be taken in consideration as a new diagnostic tool for NB. Validation in prospective clinical studies in comparison to urinary catecholamines and metanephrines is warranted., (Copyright © 2019 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: Plasma or urinary metanephrines are recommended for screening of pheochromocytomas and paragangliomas (PPGLs). Measurements of urinary free rather than deconjugated metanephrines and additional measurements of methoxytyramine represent other developments. For all measurements there is need for reference intervals., Methods: Plasma free, urinary free and urinary deconjugated O-methylated catecholamine metabolites were measured by LC-MS/MS in specimens from 590 hypertensives and normotensives. Reference intervals were optimized using data from 2,056 patients tested for PPGLs., Results: Multivariate analyses, correcting for age and body surface area, indicated higher plasma and urinary metanephrine in males than females and sex differences in urinary normetanephrine and free methoxytyramine that largely reflected body size variation. There were positive associations of age with plasma metabolites, but negative relationships with urinary free metanephrine and methoxytyramine. Plasma and urinary normetanephrine were higher in hypertensives than normotensives, but differences were small. Optimization of reference intervals using the data from patients tested for PPGLs indicated that age was the most important consideration for plasma normetanephrine and sex most practical for urinary metabolites., Conclusion: This study clarifies impacts of demographic and anthropometric variables on catecholamine metabolites, verifies use of age-specific reference intervals for plasma normetanephrine and establishes sex-specific reference intervals for urinary metabolites., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Masked uncontrolled hypertension (MUCH) is defined as controlled automated office blood pressure (BP; AOBP <135/85 mm Hg) in-clinic in patients receiving antihypertensive medication(s) but uncontrolled BP out-of-clinic by 24-hour ambulatory BP monitoring (ABPM; awake ≥135/85 mm Hg). We hypothesized that MUCH patients have greater out-of-clinic sympathetic activity compared with true controlled hypertensives. Patients being treated for hypertension were prospectively recruited after 3 or more consecutive clinic visits. All patients were evaluated by in-clinic automated office BP, plasma catecholamines, and spot-urine/plasma metanephrines. In addition, out-of-clinic 24-hour ABPM, 24-hour urinary for catecholamines and metanephrines was done. Out of 237 patients recruited, 169 patients had controlled in-clinic BP of which 156 patients had completed ABPM. Seventy-four were true controlled hypertensives, that is controlled by clinic automated office BP and by out-of-clinic ABPM. The remaining 82 were controlled by clinic automated office BP, but uncontrolled during out-of-clinic ABPM, indicative of MUCH. After exclusion of 4 patients because of inadequate or lack of 24-hour urinary collections, 72 true controlled hypertensive and 80 MUCH patients were analyzed. MUCH patients had significantly higher out-of-clinic BP variability and lower heart rate variability compared with true controlled hypertensives, as well as higher levels of out-of-clinic urinary catecholamines and metanephrines levels consistent with higher out-of-clinic sympathetic activity. In contrast, there was no difference in in-clinic plasma catecholamines and spot-urine/plasma levels of metanephrines between the 2 groups, consistent with similar levels of sympathetic activity while in clinic. MUCH patients have evidence of heightened out-of-clinic sympathetic activity compared with true controlled hypertensives, which may contribute to the development of MUCH.

Background: Measurements of plasma or urinary metanephrines are recommended for diagnosis of pheochromocytoma and paraganglioma (PPGL). What test offers optimal diagnostic accuracy for patients at high and low risk of disease, whether urinary free metanephrines offer advantages over deconjugated metanephrines, and what advantages are offered by including methoxytyramine in panels all remain unclear., Methods: A population of 2056 patients with suspected PPGLs underwent prospective screening for disease using mass spectrometric-based measurements of plasma free, urinary deconjugated, and urinary free metanephrines and methoxytyramine. PPGLs were confirmed in 236 patients and were excluded in others on follow-up evaluation., Results: Measurements of plasma free metabolites offered higher ( P < 0.01) diagnostic sensitivity (97.9%) than urinary free (93.4%) and deconjugated (92.9%) metabolites at identical specificities for plasma and urinary free metabolites (94.2%) but at a lower ( P < 0.005) specificity for deconjugated metabolites (92.1%). The addition of methoxytyramine offered little value for urinary panels but provided higher ( P < 0.005) diagnostic performance for plasma measurements than either urinary panel according to areas under ROC curves (0.991 vs 0.972 and 0.964). Diagnostic performance of urinary and plasma tests was similar for patients at low risk of disease, whereas plasma measurements were superior to both urinary panels for high-risk patients., Conclusions: Diagnosis of PPGLs using plasma or urinary free metabolites provides advantages of fewer false-positive results compared with commonly measured deconjugated metabolites. The plasma panel offers better diagnostic performance than either urinary panel for patients at high risk of disease and, with appropriate preanalytics, provides the test of choice. Measurements of methoxytyramine in urine show limited diagnostic utility compared with plasma., (© 2018 American Association for Clinical Chemistry.)

Catecholamines can be used to evaluate neuroendocrine tumors, stress, and potentially pain, but catecholamines degrade rapidly. Their metabolites normetanephrine (NME) and metanephrine (ME) have better stability in urine. In cats, urine sampling in a home environment would be beneficial to reduce effects of clinical stress and simplify sampling. We evaluated a human urine ELISA for analysis of NME and ME in feline urine, and investigated the effects of acidification, cat tray pellets, and storage time at room temperature up to 8.5 h. In 26 feline urine samples, mean NME concentration was 192 ± 80 ng/mL, mean intra- and inter-assay CV was 6.5% and 4.2%, respectively, and spike recovery was 98-101%, but dilutional recovery was unsatisfactory. For ME, mean intra- and inter-assay CV was 10.2% and 4.1%, respectively. Mean urine ME concentration was 32.1 ± 18.3 ng/mL, close to the kit's lowest standard, and spike recovery was 65-90%; the ELISA could not be validated for ME. The stability study, performed for NME on 12 urine samples, did not identify differences between acidified and non-acidified samples, cat tray pellets, or storage time, and no interaction effects. The ME ELISA was not suitable for feline urine; performance of the NME ELISA was acceptable, except for dilution recovery. For analysis of NME, feline urine can be sampled at home using cat tray pellets and stored at room temperature up to 8.5 h without acidification.

Background Plasma-free metanephrines (PFM) or urinary fractionated metanephrines (UFM) are the preferred biochemical tests for the diagnosis of phaeochromocytoma and paraganglioma (PPGL). Borderline increased results should be followed up to either exclude or confirm diagnosis. Methods We extracted all PFM and UFM results reported by our laboratory over a six-month period from the laboratory information system. We categorized patients with borderline increased results according to whether follow-up testing had been performed as suggested in the initial laboratory report. Questionnaires were then sent to all requesting doctors and medical notes reviewed where available. Results Two hundred and four patients with borderline increased PFM or UFM were identified. Sixty-five (38.5%) of 169 patients with borderline increased PFM had a repeat test out of which 36 were normal and 29 did not normalize. Of 35 patients with borderline increased UFM, 17 (48.6%) had subsequent PFM measurement, out of which 15 were normal. Questionnaires were returned to 106 (52%) patients. Of these, the most frequent indication for testing was hypertension ( n = 50); 15 patients had an incidental adrenal mass and two of these patients were diagnosed with a phaeochromocytoma. Conclusion Only 38% of patients with borderline increased PFM had a repeat PFM measurement. This was not significantly higher when compared with the 28% in a previous audit that we reported in 2010 ( P = 0.10). Forty-nine per cent of patients with a borderline increased UFM had a repeat UFM or PFM measurement. There remains a substantial possibility of missed detection of PPGL.

Pheochromocytoma and paraganglioma are neuroendocrine tumors characterized by a catecholamine production potential. The biochemical diagnosis for this type of tumor is carried out through the metanephrine titration on 24-hours urines. Some authors have suggested that the sensitivity of the test could be improved by sampling and analyzing urines 3 days in a row (cycle) versus a unique measurement but this method has never been fully evaluated. The goal of this study was to establish a comparison of diagnosis performances between urinary metanephrines measurement for 3 consecutive days, and metanephrines measurement on a unique 24-hour sample. Patients of Brest Regional University Hospital whose 3-consecutive day 24-hour urine samples had been analyzed from January 2011 to May 2017 were included in this study. The primary endpoint was the comparison of diagnostic performances of urinary metanephrine titration over a single day versus 3 consecutive days. Eighty-two patients for a total of 103 cycles among which 7 revealed a pheochromocytoma were analyzed. ROC curve analysis shows that the metanephrine cycle titration method is more efficient than the metanephrine single titration method (metanephrine: AUC=0.881 against 0.826 respectively; normetanephrine: AUC=0.946 against 0.901 respectively). Urinary titration over 3 days allows the diagnosis of 100% (7/7) of pheochromocytomas against 85.7% (6/7) for the the single urinary titration. In conclusion, metanephrine titration over 3 consecutive days of 24-hours urine samples shows a better sensitivity and better diagnosis performances for detecting pheochromocytoma and paraganglioma than the single titration method.

Background: The metanephrines (MNs) in plasma and urine were proposed as biomarkers for the diagnosis of pheochromocytomas and paragangliomas (PPGLs). However, plasma free MNs and 24h urinary fractionated MNs were not satisfactory enough in specificity for the diagnosis of PPGLs. Moreover, the collection of 24h urine was inconvenient. This work examined the diagnostic and prognostic efficiency of free MNs in spot urine for PPGLs., Methods: We measured free MNs concentration in spot urine and plasma of 28 PPGLs patients and 155 control subjects by HPLC with electrochemical detection. Postoperative free MNs levels in spot urine and plasma of 14 PPGLs patients were also determined. Creatinine (Cr) concentration was used for the correction of urine volume., Results: The specificity of spot urinary free MNs/Cr in the diagnosis of PPGLs was significantly higher than that of plasma free MNs [normetanephrine (NMN), 98.7% (95.4%-99.8%) vs 93.0% (87.4%-96.6%); metanephrine (MN), 93.6% (88.5%-96.9%) vs 84.5% (77.5%-90.0%)]. Meanwhile, the positive likelihood ratios for spot urinary free NMN/Cr and MN/Cr were 69.21 and 13.29, compared with 12.68 and 5.30 for plasma free NMN and MN, respectively. For the PPGLs patients underwent surgery, the plasma free MNs level appeared an abnormal elevation and yielded false-positive results for some patients. Our findings were validated in an independent cohort, resulting in the specificity of 100% for both urinary free NMN/Cr and MN/Cr, and 97.3% and 83.8% for plasma free NMN and MN, respectively., Conclusions: Spot urinary free MNs/Cr, superior to plasma free MNs, presented a promising biomarker for the diagnosis and prognosis of PPGLs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: Some cases of pseudopheochromocytoma have been described among hypertensive patients with obstructive sleep apnea (OSA). This study examined whether a pathological rise of urinary metanephrines is a common feature in hypertensive OSA patients and, in such a case, whether the ventilation treatment during sleep (continuous or biphasic positive airway pressure) may normalize high metanephrines levels., Methods: Patients with endocrine diseases, drug abuse, therapy with TCA and cardiovascular events in the previous 6 months were excluded. Thirty-four hypertensive patients with OSA (BMI 40.6 ± 8.7 kg/m(2)) performed three 24-h urine collections for metanephrine assessment, before and after 1 month of ventilation therapy., Results: Urinary normetanephrine (uNMT) was above the normal limit in 21 of 34 of the patients. In the 16 to 21 patients with high uNMT who were compliant to ventilation treatment, uNMT decreased in 13 by 26% and normalized in six of 13. uNMT levels were associated with apnea hypopnea index (AHI) (r = 0.799, P < 0.0001) and minimal SaO2 (r = -0.700, P < 0.01). The ventilation therapy-induced changes in AHI were associated with those in uNMT (r = 0.689, P < 0.005). In the multivariate analysis with uNMT changes as dependent variable and changes in AHI, BMI, SBP as independent variables, only AHI changes were independently associated with uNMT changes (β = 0.738, P < 0.01)., Conclusion: Two-thirds of OSA hypertensive patients have uNMT values above the normal limit. The early identification of these patients is important as ventilation therapy can correct the pathological sympathoadrenal activation. Patients who do not normalize uNMT with ventilation therapy deserve a strict follow-up as this lack of normalization may indicate insufficient ventilation therapy or resistance of sympathetic hyperactivity to this treatment, not excluding an early stage of a chromaffin tumor.

The measurement of urine catecholamine and metanephrine concentrations is important for biochemical screening and diagnosis of pheochromocytoma. The goal of this work was to develop a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determining catecholamines and metanephrines in urine to replace an existing liquid chromatographic method using electrochemical detection. Urine samples were prepared using Oasis weak-cation-exchange cartridges. The eluate was analyzed on an Agilent ZORBAX Eclipse Plus Phenyl-Hexyl column in 3 min. Adrenaline, noradrenaline, dopamine, metanephrine, normetanephrine, and their deuterated internal standards were monitored in positive electrospray ionization mode by multiple reaction monitoring (MRM). No evidence of ion suppression was observed. The assay was linear up to 5μmol/L for adrenaline, 5μmol/L for noradrenaline, 6.1μmol/L for dopamine, 5.6μmol/L for metanephrine, and 34.6μmol/L for normetanephrine, with lower limits of quantification of 5, 5, 12, 6 and 7 nmol/L, respectively. The intra-day and inter-day precisions for all analytes ranged from 0.59% to 4.64% and 1.98% to 4.80%, respectively. External quality assurance samples were assayed and showed excellent agreement with the target values. This simple method provides an improved assay for determining urine catecholamines and metanephrines.

Metanephrines (MNs) were suggested as a potential first-line biochemical index for the diagnosis of phaeochromocytomas (PHEO). In this study, we developed a simple electrochemical method for the quantitative measurement of MNs in spot urine samples. As MNs contain a hydroxyphenyl group, they could be oxidized at a certain potential to quinines, which could be further detected by the differential pulse voltammetry (DPV) method using unmodified screen-printed carbon electrode (SPCE). Meanwhile, the solid phase extraction (SPE) technique was used to eliminate the matrix effect in the samples. Consequently, free MNs from the extracted urine sample were screened in a linear range from 0.25 mg/L to 12.5 mg/L. The lowest limit of quantification (LLOQ) for MNs was 0.25 mg/L and the limit of detection (LOD) was 0.05 mg/L. Both the precisions and recoveries were sufficient for clinical applications. The urine samples from 22 patients with PHEO and 63 controls were analyzed by the proposed method. The area under the ROC curve was 0.981 (95% CI, 0.958-1.000) with the sensitivity of 95.5% and the specificity of 92.4% at the cut-off value of 0.404 mg/L in these urine samples. Overall, the proposed method provides a cost-effective, rapid and simple tool for clinical diagnosis of PHEO.

Background: Autoimmune voltage-gated potassium channelopathies have been associated with a range of neurological presenting symptoms, including central, peripheral, and autonomic dysfunction., Patient Description: We describe a 12-year-old boy who presented with nine months of pain, anxiety, and 30-pound weight loss. He was admitted for failure to thrive, then noted to be persistently hypertensive and tachycardic. Plasma metanephrines and urine metanephrines and catecholamines were elevated. Extensive investigation for causes of elevated catecholamines, such as hyperthyroidism or catecholamine-secreting tumor, was negative. A paraneoplastic panel was positive for voltage-gated potassium channel antibodies. Treatment with intravenous immunoglobulin and pulse methylprednisolone led to complete resolution of symptoms, weight gain, and normalization of vital signs and plasma metanephrines., Conclusion: Voltage-gated potassium channel antibodies should be considered as part of the differential in patients presenting with elevated metanephrine and catecholamine secretion., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background After adrenalectomy, urinary fractionated metanephrine concentrations are expected to be reduced. However, there are few studies suggesting cut-offs for adrenalectomy patients. Methods Urinary metanephrine and normetanephrine concentrations in adrenalectomy patients and two controls were compared and hormonal concentrations were evaluated via time intervals after surgery. Results The median urinary metanephrine level after unilateral adrenalectomy was lower than that of the non-pheochromocytoma controls but comparable to healthy controls. Urinary normetanephrine concentrations did not differ between adrenalectomy patients and non-pheochromocytoma controls, although both group had levels higher than those of healthy controls. The median urinary normetanephrine level in the immediate postoperative period was higher than in the later period. Conclusions Urinary metanephrine concentrations were lower after adrenalectomy, but urinary normetanephrine concentrations were not changed compared with the non-pheochromocytoma controls. However, urinary normetanephrine concentrations in the patient group were higher than levels in the heathy controls.

The development of advanced imaging techniques has increased the detection of subclinical pheochromocytomas. Because of the substantial proportions of subclinical pheochromocytomas, measurement of urine metanephrine concentrations is crucial due to detect or exclude pheochromocytoma. Although urine metanephrines are elevated in symptomatic subjects, diagnostic cut-offs according to the presence of adrenergic symptoms have not been studied. Pheochromocytomas patients who underwent adrenalectomy at Samsung Medical Center and a control group were compared to determine cut-off concentrations of urine metanephrines. An independent population was analyzed for urine metanephrines with different kits to validate the improvement in diagnostic accuracy using adjusted cut-offs. Symptom-dependent cut-offs of urine metanephrines were higher for symptomatic patients (307 μg/day in males, 235 μg/day in females for urine metanephrine, and 1,045 μg/day in males and 457 μg/day in females for urine normetanephrine) than for asymptomatic patients (206 μg/day in males, 199 μg/day in females for urine metanephrine, and 489 μg/day in males and 442 μg/day in females for urine normetanephrine). Symptom-dependent cut-offs of urine metanephrines improved a specificity from 92.7 % to 96.3 % and a high sensitivity of 97.8 % was maintained. Using the Symptom-dependent cut-offs raised diagnostic accuracy by 5.5 % (p <0.001). Similar trend was also observed in an independent population using different hormone kits. Using symptom-dependent cut-offs of urine metanephrines in symptomatic patients for pheochromocytomas resulted in a significant improvement in diagnostic accuracy in two separate cohorts.

It remains a challenge to simultaneously quantify catecholamines and metanephrines in a simple, sensitive and cost-effective manner due to pre-analytical and analytical constraints. Herein, we describe such a method consisting of a miniaturized sample preparation and selective LC-MS/MS detection by the use of second morning spot urine samples. Ten microliters of second morning urine sample were subjected to solid phase extraction on an Oasis HLB microplate upon complexation with phenylboronic acid. The analytes were well-resolved on a Luna PFP column followed by tandem mass spectrometric detection. Full validation and suitability of spot urine sampling and biological variation were investigated. The extraction recovery and matrix effect are 74.1-97.3% and 84.1-119.0%, respectively. The linearity range is 2.5-500, 0.5-500, 2.5-1250, 2.5-1250 and 0.5-1250ng/mL for norepinephrine, epinephrine, dopamine, normetanephrine and metanephrine, respectively. The intra- and inter-assay imprecisions are ≤9.4% for spiked quality control samples, and the respective recoveries are 97.2-112.5% and 95.9-104.0%. The Deming regression slope is 0.90-1.08, and the mean Bland-Altman percentage difference is from -3.29 to 11.85 between a published and proposed method (n=50). A correlation observed for the spot and 24h urine collections is significant (n=20, p<0.0001, r: 0.84-0.95, slope: 0.61-0.98). No statistical differences are found in day-to-day biological variability (n=20). Reference intervals are established for an apparently healthy population (n=88). The developed method, being practical, sensitive, reliable and cost-effective, is expected to set a new stage for routine testing, basic research and clinical applications., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objectives: Urinary catecholamines and metanephrines are biochemical indicators of pheochromocytoma. We developed and validated a rapid and precise analytical method based on solid-phase extraction (SPE) and liquid chromatography separation coupled to tandem mass spectrometry (LC-MS/MS) for measuring urinary free catecholamines and metanephrines in a clinical setting., Methods: Following SPE purification of catecholamines and metanephrines from urine specimens, chromatographic separation and quantitative detection were performed using LC-MS/MS. The developed method for simultaneous measurement of urinary free catecholamines and metanephrines was validated with clinical urine specimens and was compared with other clinical and biochemical results, including urinary total metanephrines, vanillylmandelic acid (VMA), and plasma free metanephrines., Results: The performance of our newly developed method for measuring urinary free epinephrine (EPI), norepinephrine (NE), dopamine (DA), metanephrine (MN), and normetanephrine (NMN), was acceptable. The recoveries and matrix effects of analytes were 61-107% and 84.5-130.7%. The linear ranges of each analyte were 3.8-2163μg/L, 7.4-2,359μg/L, 5.4-2,825μg/L, 3.5-2,466μg/L, and 3.7-2,569μg/L, and the coefficients of variation (CV) were less than 10% with respect to imprecision. Carryover and sample stability were also validated. Validation using clinical urine specimens by comparison with various biochemical results showed that urinary free metanephrines had comparable sensitivity (100%) and superior specificity (97.1%) to urinary total and plasma free metanephrines., Conclusions: The facile and reliable simultaneous measurement method for urinary free catecholamines and metanephrines using LC-MS/MS developed in this study is helpful in obtaining information about multiple metabolites and is applicable to routine clinical settings for the screening of pheochromocytoma., (Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Measuring urinary metanephrines aides in the diagnosis of pheochromocytomas-catecholamine producing tumors. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) allows for greater sensitivity and simpler sample preparation as compared with other techniques. Here we describe a simple LC-MS/MS method for measuring metanephrines in urine. Each urine sample was treated with diphenylboronic acid to create boronate complexes, and then applied to a Bond-Elut Plexa cartridge. After solid phase extraction, samples were concentrated and analyzed on an Atlantis T3 column with chromatographic run time totaling 8.5 min. MS/MS was set in positive electrospray ionization mode with multiple reaction monitoring for data collection. The assay was linear from 0.2 to 27.4 μmol/L and 0.3 to 14.6 μmol/L for metanephrine and normetanephrine, respectively. Intra-assay and total precision at three concentration levels over 10 days were <5 % for metanephrine and <10 % for normetanephrine.

Objective: The diagnosis of pheochromocytoma/paraganglioma (PPGL) involves detection of elevated levels of plasma and/or 24-h urine catecholamines and/or their metabolites, including metanephrines. Although these tests are reasonably sensitive, false-positive results are often encountered. Follow-up tests can provide additional information to correctly diagnose PPGL. In this regard, the utility of the urinary clonidine suppression test (UCST) remains unknown., Methods: To assess the diagnostic accuracy of the UCST in confirming or excluding PPGL, we conducted a retrospective analysis of all patients who underwent a UCST between 2000 and 2013 (n = 59; 15 PPGLs) at a single centre. Twelve-hour urine catecholamines and metanephrines were assessed before and after clonidine administration, and examined in relation to final diagnosis, PPGL or non-PPGL. Receiver operating characteristic analyses were used to identify optimal positivity cut-offs. Sensitivity, specificity, positive and negative predictive values were calculated., Results: Clonidine significantly decreased urine creatinine-corrected norepinephrine and normetanephrine in patients without PPGL (P < 0.001 pairwise) but not in patients with PPGL. Epinephrine and metanephrine levels were not significantly reduced in either group. Receiver operating characteristic (ROC) area under the curve was 0.955 [95% confidence interval (95% CI) 0.906-1.000, P < 0.001] and 0.823 (95% CI 0.706-0.940, P < 0.001) for norepinephrine and normetanephrine, respectively. Optimal cut-offs were established at 50 and 15% reductions in norepinephrine and normetanephrine, respectively, which provided high sensitivities (93.3% for both) and negative predictive values (97.4 and 96.3%). When both were concordant, higher diagnostic accuracy was achieved (100% sensitivity, 92.0% specificity). Results were similar in subgroups of individuals with borderline initial testing (n = 40) or on interfering drugs (n = 25)., Conclusion: The UCST appears to be a highly accurate test for PPGL. Further prospective studies are needed to validate these results before routine use is encouraged.

The article considers the technique of simultaneous detection of free catecholamines and free metanephrines in urine using inverse phase highly effective liquid chromatography with fluorimetric detection. The solid phase extraction was implemented on cartridges with 30 mg of hyper cross-linked polystyrene (Purosep-200). The simplicity, reproducibility and sufficient sensitivity of technique permit applying it in clinical practice to diagnose pheochromocytoma.

The aim of this study was to determine the diagnostic efficacy of free metanephrines in plasma samples drawn in the seated position compared with 24-h urinary metanephrines in detecting pheochromocytomas in Asian patients. This prospective study was conducted at Samsung Medical Center between May 2010 and July 2011. The study contained 245 subjects, including 28 patients with histologically-proven pheochromocytoma, 44 with histologically-proven non-pheochromocytoma, 112 controls suspected of having tumors but with negative investigations during two or more years of follow-up, and 45 healthy normotensive volunteers. Plasma-free metanephrines were measured by LC-MS/MS. The cut-off values with optimal sensitivity and specificity for plasma metanephrine and plasma normetanephrine were 0.33 nmol/L and 0.61 nmol/L, respectively. Both the plasma metanephrines measurement and urinary metanephrines measurement had a sensitivity of 96.4% (p = 1.00). However, the urinary metanephrines measurement was significantly more specific than the plasma metanephrines measurement (94.2% vs. 75.6%; p < 0.001). When we applied cut-off values based on BMI, specificity improved from 75.6% to 87.2%, with a comparable gain in sensitivity. From a diagnostic perspective, measurement of free metanephrines in plasma drawn in the seated position is highly sensitive but insufficiently specific when compared with measurement of 24-h urinary fractionated metanephrines. The specificity may be improved by applying cut-off values based on BMI. We suggest that free metanephrines in plasma drawn from seated position can also be used as an initial screening test to ensure that pheochromocytomas are not missed in Asian patients.

Ambulatory blood pressure variability (ABPV) is a predictor of cardiovascular risk in hypertensives. Factors that contribute to ABPV are not well described. This pilot study aimed to investigate the relationship between ABPV and urine catecholamine metabolites in order to rationalise further therapeutic intervention in the management of hypertension. Twenty individuals (14 female), aged from 23 to 71 years, with primary hypertension were identified from referrals to a regional hypertension clinic. Individuals had a 24-h ambulatory blood pressure recording (Del Mar Reynolds), and a 24-h urine collection was analysed for metadrenaline and normetadrenaline by reverse-phase chromatography (Spherisorb ODS2) and electrochemical detection (Coulochem II). Normetadrenaline concentration correlated with systolic blood pressure (SBP) variability, r=0.493, P=0.02, and with SBP coefficient of variability, r=0.490, P=0.02. Metadrenaline concentration did not correlate with either SBP variability, r=0.177, P=0.43, or coefficient of variability, r=0.185, P=0.42. A correlation was observed between 24 h urinary normetadrenaline and measures of SBP variability but not 24 h urinary metadrenaline and blood pressure variability. It is hypothesised that sympathetic activity may influence SBP variability.

This work discusses the clinical performance of deconjugated metanephrine (MN), normetanephrine (NMN) and 3-methoxytyramine (3MT) determined in the basal first morning urine using a chromatographic method with electrochemical detection for the clinical diagnosis of pheochromocytoma (PHEO) and paraganglioma (PGL). Urine samples were collected from 44 patients (36 with PHEO, 8 with PGL) aged 54+/-17 (20-78) years (22 females, 22 males). A sampling of biological materials was performed preoperatively and about one week, six months and one year after adrenal gland surgery. The control group consisted of 34 PHEO/PGL patients more than 4 months after adrenal gland surgery. All subjects in the control group were without a diagnosis of PHEO or PGL. Clinical sensitivity was 55 % for MN, 64 % for NMN, 80 % for combination of both MN and NMN, and only 23 % for 3TM. Clinical specificity calculated from the control group was 93 % for MN, 95 % for NMN, 95 % for the combination MN and NMN, and 97 % for 3TM. Cut-off values for deconjugated metanephrines in the basal urine were 310 (MN), 690 (NMN) and 250 microg/l (3MT). Chromatographic determination of deconjugated urinary metanephrines, which is simple without the necessity of special laboratory material, can serve for the screening of PHEO or PGL patients. Urine NMN and 3MT exerts an association to malignity, and all markers are associated with tumor mass. However, the principal laboratory diagnosis of PHEO or PGL must be based on plasma-free metanephrines and plasma chromogranin A with better performance in the laboratory diagnosis of PHEO or PGL.

The purpose of this study was to assess the diagnostic potential of urinary metanephrines and 3-methoxytyramine compared to urinary catecholamine determination in diagnosing antemortem cold exposure and fatal hypothermia. 83 cases of fatal hypothermia and 144 control cases were included in this study. Catecholamines (adrenaline, noradrenaline and dopamine), metanephrines (metanephrine, normetanephrine) and 3-methoxytyramine were measured in urine collected during autopsy. All tested analytes were significantly higher in hypothermia cases compared to control subjects and displayed a generally satisfying discriminative value, thus indicating urinary catecholamines and their metabolites as reliable markers of cold-related stress and hypothermia related-deaths. Metanephrine and adrenaline had the best discriminative value between hypothermia and control cases compared to other tested analytes, though with different sensitivity and specificity. These can therefore be considered the most suitable markers of cold-related stress.

Background: The purpose of this case-control study is to investigate the association between stress and periodontitis by determining stress biomarkers in saliva and urine and to determine whether oral hygiene, gingival inflammation, and tooth loss are correlated with stress biomarkers in patients with periodontitis., Methods: A total of 77 patients (41 cases [with periodontitis] and 36 controls) participated in this study. Periodontal examination findings included probing depth, clinical attachment loss, bleeding on probing (BOP), plaque index (PI), and tooth loss. Secretory immunoglobulin (sIg)A and cortisol were determined in saliva. Cortisol, creatinine-adjusted cortisol, metanephrine, normetanephrine, and total metanephrines were measured in urine., Results: Urinary metanephrine (P = 0.013) and total metanephrine (P = 0.023) levels were higher in the case group. In cases, salivary cortisol was correlated with PI (r = 0.464, P <0.01), BOP (r = 0.401, P <0.05), and tooth loss (r = 0.245, P <0.05). Urinary metanephrine levels above the median were associated with a 3.4-fold higher risk of periodontitis (95% confidence interval [CI] = 1.1 to 10.2; P = 0.029), with an 82% increase in risk for each increment of 0.05 µg/24 hours. Urinary total metanephrine levels above the median were associated with a five-fold higher risk of periodontitis (95% CI = 1.6 to 15.7; P = 0.006)., Conclusions: The present results offer new evidence of the association between urinary concentrations of catecholamine metabolites (metanephrine and total metanephrines) and chronic periodontitis. Salivary IgA level showed no statistical difference between the cases and controls. Salivary cortisol levels in the patients with periodontitis were correlated with worse PI, higher gingival inflammation, and greater tooth loss.