5 182

M Peitzsch and others Diagnosis of 182:5 499–509 Clinical Study

Overnight/first-morning urine free and methoxytyramine for diagnosis of pheochromocytoma and paraganglioma: is this an option?

Mirko Peitzsch1, Denise Kaden1, Christina Pamporaki2, Katharina Langton2, Georgiana Constantinescu2, Catleen Conrad1, Stephanie Fliedner3, Richard O Sinnott4, Aleksander Prejbisz5, Roland Därr6, Jacques W M Lenders2,7, Michael Bursztyn8 and Graeme Eisenhofer1,2

1Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany, 2Department of Medicine III, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany, 3First Department of Medicine, University Medical Center Schleswig-Holstein, Lübeck, Germany, 4School of Computing and Information Systems, University of Melbourne, Melbourne, Australia, 5Department of Hypertension, Correspondence Institute of Cardiology, Warsaw, Poland, 6Department of Medicine IV, Faculty of Medicine, University of Freiburg, should be addressed Freiburg, Germany, 7Department of Internal Medicine, Radboud University Medical Centre, Nijmegen, The to G Eisenhofer Netherlands, and 8Department of Medicine, Hypertension Unit, Hadassah-Hebrew University Medical Center, Email Jerusalem, Israel graeme.eisenhofer@ uniklinikum-dresden.de

Abstract

Objective: Sympathoadrenal activity is decreased during overnight rest. This study assessed whether urinary-free , 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,

European Journal of Endocrinology 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. 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 , 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 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.

European Journal of Endocrinology (2020) 182, 499–509

https://eje.bioscientifica.com © 2020 European Society of Endocrinology Published by Bioscientifica Ltd. https://doi.org/10.1530/EJE-19-1016 Printed in Great Britain Downloaded from Bioscientifica.com at 09/28/2021 05:56:35AM via free access

-19-1016 European Journal of Endocrinology https://eje.bioscientifica.com sympathoadrenal activitymay potentiallybeavoidedby For urine collections, daily influences that increase minimized byanovernight fastbeforebloodsampling. influencescanbe results. Fortheplasmatest, dietary normetanephrine ( methoxytyramineanddeconjugated and urinary stressors. Influencesofdiet,whichcanincreaseplasma in sympathoadrenalactivityassociatedwithdailylife urine collectionsalsodonoteasilycontrolforincreases incomplete orincorrectcollections.Twenty-four-hour for patients and also susceptible to inaccuracy due to to 24-hurinecollections.Thelatterareinconvenient and morerapidprocedureofbloodsamplingcompared tests ( is littleifanydiagnosticadvantageofplasmaoverurine supine rest ( particular, bloodsamplesshouldbecollectedafter is avoidedduringdiagnostictestingforPPGLs;in of their metabolites, it is important that suchactivation increases secretionofcatecholaminesandproduction urine, whetherfreeordeconjugated. useful in plasma, exhibits limited diagnostic utility in O-methylated metaboliteofdopamine,whichalthough included measurementsofmethoxytyramine,the study involvingover2000patients( metanephrines hassincebeenestablishedinaprospective metanephrines comparedtobothplasmaandurinary-free deconjugated Inferior diagnosticaccuracyofurinary measurements of the deconjugated metabolites inurine. in plasmaprovidedsuperiordiagnosticaccuracyover not clearwhethermeasurementsofthefreemetabolites publication oftheEndocrineSocietyGuidelines( for plasmameasurements( to thedeconjugated metabolites hasbeen confirmed diagnostic accuracyofthefreemetanephrinescompared the bodyfromfreemetanephrines( conjugated metabolites produced at different sites in deconjugation stepandlargelyrepresentsulfate- however, commonly measured after an acid-hydrolysis other metabolites( for diagnosisovertheparentcatecholaminesand tumor derivativesandtherebytheirsuperioraccuracy specific sources fromwithinchromaffin cellsand and paraganglioma(PPGL),recognizingtheirrelatively and metanephrine)fordiagnosisofpheochromocytoma orplasmametanephrines(i.e.normetanephrine urinary The EndocrineSociety(USA)guidelinesrecommend Introduction Clinical Study As theactivationofsympathoadrenalsystems 6 ); the only remaining advantage is thesimpler 5 , 6 , 7 9 , ), canalso contribute tofalse-positive 8 1 ). Without such precautions, there ). Urinary metanephrinesare, ). Urinary 3 ). However, atthetimeof M Peitzschandothers 4 ). Thatstudyalso 2 ). Superior 1 ), itwas on functional imaging evidence (e.g. for metastatic examination ofresectedtumors, butoccasionallydepended Confirmation usually involved histopathological function and thereby provide reference comparators for verify impactsofovernightrestonsympathoadrenal catecholamineswerealsomeasuredto rest. Urinary daytime wakinghoursandthesecondduringovernight were splitintotwocollectionperiods,thefirstduring ( prospective monoamine-producing tumor (PMT)study was addressedinthepresentstudy, asub-studyofthe to measurementsin24-hcollections.Thishypothesis diagnosis of PPGLs and improved accuracy compared would offersimilarperformancetotheplasmatestfor and methoxytyramineinovernightcollectionsofurine hypothesized thatmeasurementsoffreemetanephrines than indaytimecollections( lower when sampled after overnight rest or during sleep outputsofnorepinephrineandepinephrineare urinary it iswell-establishedthatplasmaconcentrationsand collecting urineafteraperiodofovernightrest.Indeed, confirmed in79andexcluded in627patients( pmt-study.pressor.org standard-operating proceduresavailableonline( to anEthicscommittee-approvedclinicalprotocoland multiple phases,fromscreeningtofollow-up,according ofPPGL.The studycontained or (4.)previoushistory findings of an abdominal or adrenal incidentaloma riskof PPGL;(3.) catecholamine excess;(2.)hereditary risk forPPGLs:(1.)signsand/orsymptomsofpresumed there werefourmaincriteriaforestablishingsuspicion or outputsofmetabolites.Inbrief, and overnighturinary were availableandconsecutivelyanalyzedfordaytime for whomseparatedayandovernighturinecollections subset of over 2000 patients enrolled into the PMT study published elsewhere( in themanuscriptandsupplementofmainstudy under a multicenter prospective clinical protocol described were enrolled into this cross-sectional diagnostic study years (range13–85)andwithsuspicionoratriskforPPGLs A totalof706patients(348males)atamedianage54 Subjects Subjects andmethods downstream impactsonthemetabolites. pheochromocytoma Diagnosis of 4 ), bythecollectionofurinesamplesover24-hthat Based ontheaforementionedconsiderations,itwas In the706patientstestedforPPGLs,tumorswere ). 4 ). These706patientsrepresenteda Downloaded fromBioscientifica.com at09/28/202105:56:35AM 10 , 11 , 12 182 , 13 :5 ). Table 1 https:// 500 via freeaccess ).

European Journal of Endocrinology tandem mass spectrometry (LC-MS/MS) according totandem mass spectrometry usingliquidchromatography- single laboratory epinephrine anddopamine) wereperformedata methoxytyramine andcatecholamines (norepinephrine, metanephrines, 3-methoxytyramine andurinary-free Analyses ofplasma-freemetanephrinesand Biochemical analyses were performed. ice,where thebiochemicalanalyses ondry laboratory study centerswereshippedtothecentralanalytical separate theplasma.Allspecimenscollectedatdifferent samples werekeptcoolat4 30 minupuntilthetimewhenbloodwasdrawn.Blood overnight fast,bymaintainingthesupinepositionfor Blood samplesweretakeninthemorninghoursafteran determined andsamplesaliquotedforstorageat on thefinaldayofcollection,atwhichtimevolumeswere outputs. Urinespecimenswerereturnedtostudycenters outputsweresummedtoprovide24-hurine urinary including thefirst-morningurine.Daytimeandovernight collections included all subsequent collections up until and before overnight recumbent rest or sleep. Overnight continuing collections until inclusion of the last void by excludingthefirst-morningurinecollectionand collections. Thiswasachievedfordaytimecollections Urine collectionsweresplitintodaytimeandovernight Urine andbloodcollections informed consentbeforestudyinclusion. described PMTsub-study. Allsubjectsprovidedwritten, four centersthatcontributedsubjectstothepresently Institute ofCardiologyatWarsaw, thesecomprisingthe the University Medical Center Schleswig-Holstein and the Hospital Dresden,theRadboudUniversityMedicalCenter, approved bytheEthicsCommitteesofUniversity under whichpatientsorvolunteerswereenrolled Theclinicalprotocols establishing referenceintervals. patient population,wasincludedforcomparisonand Dresden andmatchedaccordingtoagesexwiththe a medianageof51years(range18–-82),recruitedat of 335normotensiveandhypertensivevolunteerswith paragangliomas in 6patients. A reference population and 17hadparagangliomas,includingheadneck the 79patientswithPPGLs,61hadpheochromocytomas criteria andproceduresdescribedpreviously( disease), whileexclusioninvolvedfollow-upaccordingto Clinical Study ° C beforecentrifugationto M Peitzschandothers 4 ). Among − 80 ° C. (SAS InstituteInc,Cary, NC).Differencesbetween Statistical analysesutilizedtheJMPsoftwarepackage Statistical analysis are establishedhere. overnight collectionsofurinenormalizedtocreatinine described elsewhere( outputsoffreemetaboliteshavebeen and 24-hurinary elevated fromnormaltestresultsforplasmametabolites usedtodistinguish Diagnostics). Thereferenceintervals were determinedonaCobas8000analyzer(Roche creatinineconcentrations (nmol/mmol). Urinary and are expressed as nmol per mmol creatinine differences indilutionaccordingtooutputsofcreatinine and overnight urine collections were normalized for in nmolperday(nmol/day),whereasthosesplit Measurements in24-hurinecollectionsareexpressed previously describedandvalidatedmethods( intervals for metanephrineandmethoxytyramineutilized intervals importance oftheother two metabolites,reference 97.5 percentiles. Incontrast,recognizingthesecondary ( population. Consistentwithpreviousconsiderations collections fromdistributionsofdataforthereference establishedhereforday andovernight reference intervals, of diagnosticsensitivityandspecificityaccording to in test performance were also assessed from comparisons utilized theJMPmodelcomparisonfunction.Differences CIs ofAUCsand95%differencesbetweenAUCs, significance ofdifferences, includingestimation of95% (AUCs) to assess the Comparisons of areas-under-curves constructedusinglogistic regression. (ROC) curves primarily assessedfromreceiver-operatingcharacteristic (95%CIs). intervals expressed asgeometricmeansand95%confidence transformation. Data from those analyses were then ratios ofovernighttodaytimecollectionsbylogarithmic assessed byparametricanalysesafterfirstnormalizing changes from daytime to overnight collections were correlation coefficient. Differencesbetween groups in between variableswasassessedusingSpearman’s rank rank sumtest,whilethesignificanceofrelationships collections wereassessedusingthepairedWilcoxon sign- analyses. Differencesbetweendaytimeandovernight test, withtheSteel–Dwasstestemployedforpost-hoc of analyteswereestablishedusingtheKruskal–Wallis outputs groups inplasmaconcentrationsandurinary pheochromocytoma Diagnosis of 17 ), uppercut-offsfornormetanephrine utilizedthe Differences intheperformanceofdiagnostictestswere 4 , Downloaded fromBioscientifica.com at09/28/202105:56:35AM 17 ), whereasthosefordayand https://eje.bioscientifica.com 182 :5 14 , 15 501 , 16 via freeaccess ). ). European Journal of Endocrinology https://eje.bioscientifica.com fold higher( outputsfordopaminewereonly1.2- overnight urinary higher outputsformetanephrine. Incontrast,daytimeand without PPGLs,comparedto4.1-and4.7-fold( 7.3-fold higher( outputsforepinephrinewererespectively 3.2-and urinary for normetanephrine( to larger9.5-and11.9-foldhigher( in patientswithPPGLsthanwithoutPPGLs,compared were respectively3.6-and3.7-foldhigher( outputs of norepinephrine Daytime and overnight urinary and metabolites Daytime andovernighturinarycatecholamines was excluded. respective metabolitesthanpatientsinwhomdisease higher ( and methoxytyramine9.3-,5.1-1.6-fold concentrations ofnormetanephrine,metanephrine showed 7.7-,4.4-and2.7-foldhigher( to pheochromocytoma.PatientswithconfirmedPPGL some patientswithpasthistoriesofadrenalectomiesdue former thanthelattergroup, this reflectinginclusionof free metanephrinewere13–14%lower( excretionofplasma concentrationsand24-hurinary sex-matched referencepopulation( were excludedandforsubjectsoftheage-matched between patients tested for PPGLs in whom tumors normetanephrine andmethoxytyraminedidnotdiffer outputs of freePlasma concentrations and 24-h urinary catecholamine metabolites Plasma and24-hurinaryoutputsof Results this article). section on Tablefrom 95%CIsofdifferences (Supplementary 1,see between testswereassessedusingMcNemar’s testand positive results.Differencesinsensitivityorspecificity negative resultsamongbothtrue-negativeandfalse- specificity wasestimatedfromthepercentage oftrue- negative resultsforpatientswithPPGLs.Diagnostic positive resultsamongbothtrue-positiveandfalse- sensitivity wasestimatedfromthepercentage oftrue- the 99.5percentiles. 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European Journal of Endocrinology https://eje.bioscientifica.com not forovernightcollections. However, measurements for daytimecollectionsof urine thanforplasma,but sensitivity formetabolites was alsolower( metanephrine andmethoxytyramine, thediagnostic for urine-freenormetanephrine and99.5percentiles for specific uppercut-offs,derivedfrom97.5percentiles ( sensitivity thantheurinetestatidenticalspecificities plasma testdemonstratedhigher( in plasmaand24-hcollectionsofurine( normetanephrine, metanephrineandmethoxytyramine metabolites. catecholamines with increased bycombiningurinary ( metabolites was higher metabolites. The AUC for urinary for tests involving 24-h and overnight collections of fall inurinarymetanephrinecomparedtoepinephrine. compared tonorepinephrine; * data areshownasgeometricmeanswithconfidenceintervals. patients inwhomPPGLswereexcludedandconfirmed.All metanephrine insubjectsofthereferencepopulationand norepinephrine, normetanephrine,epinephrineand Percent day-to-nightdecreasesinurinaryoutputsoffree Figure 1 Table 3 P P

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= 0.0219) specificitythanboth 182 :5 P =0.0253) 504 via freeaccess European Journal of Endocrinology methoxytyramine inovernight orfirst-morningurine Although measurementsof freemetanephrinesand Discussion daytime collections. specificity washigher( differ between overnight and daytime urinecollections, at theseoptimizedcut-offs,althoughsensitivitydidnot urine collectionsatanearequivalentspecificity. Moreover overnight collections was higher ( for plasmameasurements.Importantly, thesensitivity for of 97.5(93.9–100.0)%andspecificity94.7(93.0–96.5)% (92.4–96.1)%, only marginally lower thanthesensitivity sensitivity of93.7(88.2–99.2)%andaspecificity94.3 in overnight urine collections yielded adiagnostic 3 combined daytime and overnight urine collections ( using sex-specific upper cut-offs derived from both of metabolites in overnight urine collections was achieved combined day-and-nightcollectionsusedtoestablishdiagnosticperformanceinTable3. a Combined Night Day Free methoxytyramine(0.5and99.5percentiles) Combined Night Day Free metanephrine(0.5and99.5percentiles) Combined Night Day Free normetanephrine(0.5and99.5percentiles) Combined Night Day Free methoxytyramine(2.5and97.5percentiles) Combined Night Day Free metanephrine(2.5and97.5percentiles) Combined Night Day Free normetanephrine(2.5and97.5percentiles) to the97.5and99.5percentilesofdistributionsfor335subjectsreferencepopulation. shown separatelyformaleandfemaleadultsinunitsofnmolmetabolitepermmolcreatinineweredeterminedaccording Table 2 Indicates dayandnightsex-specificuppercut-offsusedtoestablishdiagnosticperformanceinTable3; ). Usingtheseuppercut-offs,measurementsofmetabolites Clinical Study Optimized diagnosticperformanceformeasurements Reference intervalsfordaytime,overnightandcombineddaynighturinefreemetabolites.are P

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= 0.0253) than for 24-h Table of norepinephrine to maintain blood pressure ( associated withincreased sympathoneuronalrelease test ( the urinary diagnostic accuracyoftheplasmatestisnobetterthan rest ( of theplasmatestrequiresbloodsamplingaftersupine when itisappreciatedthatthehighdiagnosticaccuracy sampling ( estimated tobenearly15-foldhigherthanforseated cost ofbloodsamplinginthesupinepositionhasbeen certain healthcaresystems,suchasinBritain,wherethe supine restperiod.Thelatterisparticularlyrelevantfor collect blood aftertherecommended 20-min minimum notfeasibleto where itiscost-prohibitiveorotherwise useful for centers where the plasma test is unavailable or for patientsassociatedwith24-hcollectionsandmaybe 24-h collections. Such analternative minimizes problems overnight urinecollectionsmayofferaviablealternativeto for diagnosisofPPGL,thisstudydoesindicatethat collections werenotasaccurateplasmameasurements 21 pheochromocytoma Diagnosis of ); thus sampling of blood in the seated, compared to The aforementioned considerations are important 5 , 6 , 7 18 , 8 ). ). Without supinerestandotherprecautions, 1.9–24.1 1.6–21.7 3.8–45.3 3.0–28.3 4.2–54.3 7.5–32.2 8.1–33.2 7.6–40.5 2.5–18.9 2.2–14.9 2.3–19.4 5.0–24.7 4.1–17.8 5.3–31.4 3.5–42.4 4.9–43.6 3.1–58.1 1.9–23.5 Females 6 ). Thisisbecauseupright posture is a a b a a a,b b b Indicates sex-specificcut-offsfrom Downloaded fromBioscientifica.com at09/28/202105:56:35AM https://eje.bioscientifica.com 182 :5 2.0–17.8 1.8–24.5 3.6–30.8 0.5–27.5 3.9–33.2 7.8– 30.3 7.9–33.3 8.2–33.3 2.9–16.1 2.1–14.2 2.7–16.1 4.5–21.0 3.3–15.9 4.8–26.2 6.6–35.9 6.9–43.2 6.2–40.9 2.0–22.9 Males a,b b a a b a a 19 505 , 20 via freeaccess , European Journal of Endocrinology https://eje.bioscientifica.com differences betweenAUC( Significance ofdifferencesin AUCareshownwith95%CIof under ROCcurves(AUC)and95% CIsofareasundercurves. catecholmines andmetabolites (B).Dataareshownwithareas catecholamines andthecombinationofovernighturinary and forovernighturinaryfreemetabolitesvs normetanephrine, metanephrineandmethoxytyramine(A) outputs for24-handovernightcollectionsoffree logistic regressionforplasmaconcentrationsvsurinary Receiver-operating characteristiccurvesconstructedusing Figure 3 Clinical Study ∆ AUC). M Peitzschandothers and acomparisongroupof166hypertensives( study involving16patientswithpheochromocytoma 28 involving sixpatientswithpheochromocytoma( proposed intworeports1975and1979,each to afull24-hisnotnew. Thisideawasoriginally excess involvingurinecollectedovernightcompared diagnostic performance for tests of catecholamine activity duringsleepmightresultinimproved anddiagnostictesting. intervals sympathoadrenal activityforbothestablishingreference is thereforeimportanttominimizeimpactsofincreased expense of compromised diagnostic sensitivity ( may minimizethefalsepositives( established fromseated than supine sampling intervals 22 normetanephrine andahighrateoffalsepositives( the supine, position results in increasedplasma-free adrenal medullary cells does provide a theoretical adrenal medullary comparedtometabolismwithin by sympatheticnerves derived frommetabolism of norepinephrinereleased contrast, the larger component of normetanephrine metanephrine, thoughsubstantialforepinephrine.In compared to24-hurinecollectionsisminimalfor from thosecells( a source thatisindependentofepinephrinesecretion cells, vesicles intothecytoplasmofadrenalmedullary from epinephrinecontinuouslyleakingstorage is explainedbythepredominantsource ofmetanephrine day-to-night decreaseinmetanephrinethanepinephrine sympathoadrenal activation( of catecholaminesandtheirO-methylatedmetabolites to with previous findings of differences in responsiveness decreases of theparentcatecholamines are consistent subjects withoutPPGLwereproportionallysmallerthan normetanephrineandmetanephrineamong urinary-free catecholamine excretion’. avoids theinfluenceofstressorposturalchangeson pheochromocytoma providesacollectionperiodthat epinephrine excretionduringsleepinpatientswithout concluded that‘suppressionofnorepinephrineand the 82%specificityfor24-hcollections.Theauthors collections, at 98%, was also improved compared to 24-h collections.Diagnosticspecificityforovernight compared to14outof16whoshowedelevationsfor norepinephrineaboveuppercut-offsovernight urinary 16 patientswithpheochromocytomahadvaluesfor pheochromocytoma Diagnosis of , ). Thesestudieswereshortlyfollowedbyanother The presentfindingsthatday-to-nightdecreasesof The conceptthatreducedsympathoadrenal 23 , 24 , 25 ). Althoughhighercut-offsofreference 31 ). Thus,anyadvantageofovernight Downloaded fromBioscientifica.com at09/28/202105:56:35AM 30 ). Inparticular, thesmaller 24 182 , 26 :5 ), thisisatthe 5 29 , 506 ). All 7 ). It 27 via freeaccess 5 , , European Journal of Endocrinology that measurementsofthefree metabolitesinovernight/ earlier studies( performed atothertimesduring theday. in first-morning urinecollectionscompared tocollections this suggeststhattheremayalsobediagnosticadvantages forboth; least whenusingthesamereferenceintervals of metabolitesinovernightthandaytimecollections, at were similarbutspecificitieshigherformeasurements urine collections( in patientswithPPGLsusingovernightcomparedto24-h a diagnosticadvantageinassessingcatecholamineexcess provides somesupportforsuggestionsthattheremaybe in overnightcomparedto24-hurinecollections.This higher atsimilarspecificitiesformeasurementsperformed diagnosticsensitivitywas chosen referenceintervals, overnight and24-hurinecollections,withappropriately performance formeasurementsoffreemetabolitesin showed similar diagnostic offs. Although ROC curves reference populationforindependentlyestablishingcut- excluded andconfirmed,aswellanappropriatelysized includes largergroupsofpatientsinwhomPPGLswere ( specificity comparedtothedeconjugatedmetabolites compared totheparentcatecholaminesandimproved which providesuperioraccuracyfordiagnosisofPPGLs metanephrines and methoxytyramine, urinary-free in earlier studies ( overnight comparedto24-hurinecollections. advantage formeasurementsofnormetanephrinein between tests,95%CIsofdifferencestestsandexact significance ofdifferencesinsensitivityandspecificitywereestablishedbypairedanalyses(McNemar’stest).SeeSupplementaryTable1for differences higher specificitythanthecorrespondingovernighttestandotherdaytimetest;*** plasma test; established usingsex-specificuniversaluppercut-offsforcombineddayandnightcollectionsasstipulatedinTable2; a Overnight urinary-freemetabolites Daytime urinary-freemetabolites Daytime andovernightstrategiesaccordingtooptimizeduniversalday/nightuppercut-offs Overnight urinary-freemetabolites Daytime urinary-freemetabolites Daytime andovernightstrategiesaccordingtorespectivedaytimeuppercut-offs 24-h urinary-freemetabolites Plasma-free metabolites Conventional testingstrategies parentheses. 95% CIsinbracketsandnumbersofpositiveornegativeresultsrelativetothesum free metabolitescomparedtodaytimeandovernighturinecollectionsnormalizedcreatinine.Dataareshownasmeanswith Table 3 Test performanceestablishedusinguppercut-offsstipulatedinTable2forbothdayandnightsex-specificreferenceintervals; 4 Clinical Study ). Compared to those earlier studies, our study also Thus, ourstudybuildsonconceptsintroduced Despite supportingtheconcepts introducedin Diagnostic testperformanceofconventionaltestingstrategiesplasma-freemetabolitesor24-hurinaryoutputs § P

< 0.05 highersensitivitythan24-hurinetest;* 27 27 , 28 27 , 28 , , 29 , 28 29 ), ourstudydoesnotestablish , ). Also,diagnosticsensitivities 29 ), but with a focus on M Peitzschandothers P < P -values. 0.05 and** P

< 93.7 (88.2–99.2)% (74/79) 93.7 (88.2–99.2)% (74/79) 93.7 (88.2–99.2)% (74/79) 89.9 (83.1–96.7)% (71/79) 87.3 (79.8–94.8)% (69/79) 97.5 (93.9–100.0)% (77/79) 0.0001, lowerspecificitythanplasmaand24-hurinetests; first-morning urinesamplesoffersimilardiagnostic sub-study was that reference intervals for day and night sub-study was that reference intervals of thePMTstudy( to plasma-freemetabolites. compared lower diagnosticaccuracyof overnighturinary and bloodpressure( or reverse day-to-night falls in sympathoadrenal activity healthier volunteers.Suchinfluencesareknowntoblunt sleep apneasyndrome,inthepatientgroupthan associated with sympathoadrenal activation, such as may reflectahigherprevalenceofpathologicalconditions (15.5% vs 6.6%) than inthe reference population. This norepinephrine (13.2%vs3.6%)andnormetanephrine excretionof increases ratherthandecreasesinurinary more than2-foldlargerproportionsofovernight that patientsinwhomPPGLswereexcludedexhibited in some patients. This possibility is suggested by findings additionally reflectnocturnalsympathoadrenalactivation in plasmacomparedtoovernighturinecollectionsmay of dopamineandmethoxytyramine. excretion to the lack of day-to-night differences in urinary circulating L-dopa( methoxytyramine inurinefromrenaldecarboxylationof superiority reflectsderivationofmostdopamineand detection ofdopamine-producingtumors( of methoxytyramineinplasmacomparedtourinefor fast. Inpart,thismayreflectsuperiorityofmeasurements collections of blood after supine rest andan overnight accuracy tomeasurementsperformedinplasmausing pheochromocytoma Diagnosis of P Apart fromalreadyoutlined strengthsandlimitations Diagnostic superiority of measurements of metabolites

< 0.0001, higherspecificitythancorrespondingdaytimetest.The Sensitivity 4 ), alimitationofthepresently described 33 11 § § § † † b , ) andmaytherebycontributetothe

34 Downloaded fromBioscientifica.com at09/28/202105:56:35AM a ), whichpotentiallycontributes 94.3 (92.4–96.1)%(591/627)*** 88.2 (85.7–90.7)%(553/627)** 87.6 (85.0–90.2)%(549/627)** 92.0 (89.9–94.1)%(577/627)* 94.7 (93.0–96.5)%(594/627) 94.7 (93.0–96.5)%(594/627) † P

https://eje.bioscientifica.com < 0.02, lowersensitivitythan 182 Specificity :5 b Test performance ¶ 4 P ,

< 32 0.002, ). This 507 ¶ via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com References the in detailed are accuracy Diagnostic supplement. Reporting for Standards STARD German Research Foundation Project number 314061271-CRC/TRR 205/1). (DFG, Forschungsgemeinschaft Deutsche the by supported was work This Funding be could that interest of conflict perceived asprejudicingtheimpartialityofthisstudy. no is there that declare authors The Declaration ofinterest EJE-19-1016 at paper the of version online the to linked is This Supplementary materials problems associatedwithincorrect24-hcollections. minimizing inconveniencetopatientsandavoiding collections asanalternativeto24-hcollections,thereby provide evidencetosupportuseofnocturnalurine performance for the various urine measurements does measuresofdiagnostic of differencesinROCcurve-based further large-scaleprospectivestudy. Nevertheless,lack of overnightcomparedto24-hcollectionsrequiresa protocol. Whetherthereisanytruediagnosticadvantage a thirdoftheover2000patientsenrolledintoPMT day andnighturinecollectionswerecarriedoutinonly on first-morningurinecollections. who shouldbeincludedinanyfurtherstudiesfocusing urine collectionsareparticularlyimportantforthisgroup collections ofurineinchildrencanbeunreliable,spot indicated byclearlypositivetestresults.Since24-h age of18,onlyonewhomhadapheochromocytoma study includedthreepatientstestedforPPGLunderthe when normalizedtocreatinine( catecholaminesandmetabolites,particularly urinary are essential for children, age-specific reference intervals urine collectionswereonlyestablishedforadults.In 2 1 Clinical Study Eisenhofer G. Freeortotalmetanephrines fordiagnosisof Lenders JWM, Duh QY, Eisenhofer G,Gimenez-Roqueplo AP, 47 pheochromocytoma: whatisthedifference? jc.2014-1498) and Metabolism Society ClinicalPracticeGuideline. Society. Pheochromocytomaandparaganglioma:anEndocrine Grebe SK, Murad MH,Naruse M,Pacak K,Young WF &Endocrine Another limitationofthepresentstudywasthatsplit 988–989. . (https://doi.org/10.1093/clinchem/47.6.988) 2014 99 1915–1942. Journal ofClinicalEndocrinology Journal (https://doi.org/10.1210/ M Peitzschandothers 35 , 36 Clinical Chemistry https://doi.org/10.1530/ , 37 ). Thepresent 2001

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