6 182

N Bäcklund and others Salivary in Cushing 182:6 569–582 Clinical Study diagnosis

Reference intervals of salivary and cortisone and their diagnostic accuracy in Cushing’s syndrome

Nils Bäcklund1, Göran Brattsand2, Marlen Israelsson2, Oskar Ragnarsson3,4, Pia Burman5, Britt Edén Engström6, Charlotte Høybye7, Katarina Berinder7, Jeanette Wahlberg8, Tommy Olsson1 and Per Dahlqvist1

1Department of Public Health and Clinical Medicine, Umeå University, Umeå, Sweden, 2Department of Medical Biosciences, Clinical Chemistry, Umeå University, Umeå, Sweden, 3Department of Internal Medicine and Clinical Nutrition, Institute of Medicine at Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, 4Department of Endocrinology, Sahlgrenska University Hospital, Gothenburg, Sweden, 5Department of Endocrinology, Skåne University Hospital, University of Lund, Malmö, Sweden, 6Department of Medical Sciences, Endocrinology and Mineral Metabolism, Uppsala University, Uppsala, Sweden, 7Department of Molecular Medicine and Surgery, Patient Area Endocrinology and Correspondence Nephrology, Inflammation and Infection Theme, Karolinska Institute and Karolinska University Hospital, Stockholm, should be addressed Sweden, and 8Department of Endocrinology, Department of Medical and Health Sciences, Linköping University, to N Bäcklund Linköping, Sweden Email [email protected]

Abstract

Objective: The challenge of diagnosing Cushing’s syndrome (CS) calls for high precision biochemical screening. This study aimed to establish robust reference intervals for, and compare the diagnostic accuracy of, salivary cortisol and cortisone in late-night samples and after a low-dose (1 mg) dexamethasone suppression test (DST). Design and methods: Saliva samples were collected at 08:00 and 23:00 h, and at 08:00 h, after a DST, from 22 patients with CS and from 155 adult reference subjects. We also collected samples at 20:00 and 22:00 h from 78 of the reference subjects. Salivary cortisol and cortisone were analysed with liquid chromatography-tandem mass spectrometry. The reference intervals were calculated as the 2.5th and 97.5th percentiles of the reference population

European Journal of Endocrinology measurements. Diagnostic accuracies of different tests were compared, based on areas under the receiver-operating characteristic curves. Results: The upper reference limits of salivary cortisol and cortisone at 23:00 h were 3.6 nmol/L and 13.5 nmol/L, respectively. Using these reference limits, CS was detected with a sensitivity (95% CI) of 90% (70–99%) and specificity of 96% (91–98%) for cortisol, and a 100% (84–100%) sensitivity and 95% (90–98%) specificity for cortisone. After DST, cortisol and cortisone upper reference limits were 0.79 nmol/L and 3.5 nmol/L, respectively. CS was detected with 95% (75–100%) sensitivity and 96% (92–99%) specificity with cortisol, and 100% (83–100%) sensitivity and 94% (89–97%) specificity with cortisone. No differences in salivary cortisol or cortisone levels were found between samples collected at 22:00 and 23:00 h. Conclusion: Salivary cortisol and cortisone in late-night samples and after DST showed high accuracy for diagnosing CS, salivary cortisone being slightly, but significantly better.

European Journal of Endocrinology (2020) 182, 569–582

https://eje.bioscientifica.com © 2020 European Society of Endocrinology Published by Bioscientifica Ltd. https://doi.org/10.1530/EJE-19-0872 Printed in Great Britain Downloaded from Bioscientifica.com at 09/25/2021 10:23:41PM via free access

-19-0872 European Journal of Endocrinology https://eje.bioscientifica.com use of LC-MS/MS increases, solid reference intervals are use ofLC-MS/MSincreases, solidreferenceintervals it has a low limit of quantification. Importantly, as the cortisolandcortisone, measurements ofsalivary immunoassays. LC-MS/MS alsoallowssimultaneous MS) hastheadvantageofhighspecificity, compared to (LC-MS/ chromatography-tandem massspectrometry DST ( accuracy forsamplescollectedbothlateatnightandafter diagnostic cut-off levels, and diagnostic reference intervals, CS withcurrentmethods,weneedtodeterminerobust cortisone fordiagnosing usefulness ofmeasuringsalivary cortisol( salivary cortisone thanwith levels correlatebetterwithsalivary Moreover, it hasbeen suggested that plasma free cortisol reduce problemsassociatedwithcontamination. cortisone,insteadofcortisol,might measuring salivary be established( remainsto although thenormalrange(referenceinterval) contamination from blood or exogenous hydrocortisone, cortisol:cortisone ratio can be used to detect salivary ratio in blood ( cortisone arepresentina1:5ratio,comparedtothe5:1 type 2(11 gland bytheenzyme11 the cortisolisconvertedintocortisoneinsalivary containing oralcontraceptives(OCs)( plasma CBG levels, for example, in womenusing venous blood sample and is affected by differences in depends onplasmacortisolanalysis,whichrequiresa dexamethasone, excludesCS.TheconventionalDST a lowplasmacortisolinthemorning,aftertakingoral (HPA)the hypothalamic-pituitary-adrenal axis;thatis, metabolism. TheDSTevaluatesthesuppressibilityof LNSC isnothamperedbyvariationsindexamethasone cortisol-binding globulin(CBG)( levels, and independence from variations in plasma procedure, closecorrelationwithfreeplasmacortisol ( cortisol (LNSC).Allofthesemethodshaveprosandcons freecortisol(UFC);orlate-nightsalivary 24-hour urinary low-dose (1mg)dexamethasonesuppressiontest(DST); biochemical screeningmethodsforCSincludeover-night of hypercortisolism. Currently, therecommended combination ofclinicalsignsandbiochemicaltests challenge in clinical endocrinology, relying on the Diagnosis ofCushing’s syndrome(CS)isstillamajor Introduction 1 ). Advantages of salivary cortisolarethesimplesampling ). Advantagesofsalivary Clinical Study Analysing salivary with liquid Analysing salivary During thepassagefrombloodtosaliva,mostof 3 , 15 , 16 β , -HSD2). Thus,insaliva,cortisoland 17 5 5 , , 3 , 8 , 6 18 , , 15 9 ). 7 , ). However, toensuretheclinical , 10 8 β -hydroxysteroid dehydrogenase , , 9 11 ). Knowledge of the normal , 12 2 N Bäcklundandothers , , 3 13 , 4 , 1 ). Furthermore,the ). 14 ). Alternatively, Participants Methods different eveningsamplingtimes. of gender, oestrogenstatus,age,tobaccouse,season,and cortisol:cortisoneratio;andtoevaluatetheeffects salivary be superiortoasingletest;establishthenormal samples collected both late at night and after DST, might also aimedtotestwhetheracombinedtest,withsalivary compare thediagnosticaccuraciesofdifferenttests.We tests forcorrectlyidentifyingpatientswithCS;andto MS; tocalculatethesensitivityandspecificityofthese 08:00 h, 23:00 h, and after DST, measured with LC-MS/ cortisolandcortisone,sampledat forsalivary intervals screening tests. needed fordeterminingthediagnosticaccuracyofCS because further investigation was required to exclude CS after DSTwereexcludedfrom thereferencepopulation, reference subjects with plasma cortisol samples ( illness duringthesamplingperiod( glucocorticoids ( subjects wereexcluded,duetouseofinhaledornasal reported menopausalstatusandoestrogenuse. insomnia severityindex(ISI)( and diurnalrhythmdisturbanceswereassessedwiththe and fastingbloodglucosemeasurements.Potentialsleep bloodsamples forhemoglobin and collectedcapillary height, weight,waistcircumference, andbloodpressure, brief health questionnaire, measured body temperature, by anexperiencedresearch nurse,whoadministered a informed consent, reference individuals were assessed ( rhythm (awakebetween24:00and05:30h),fever medication duringtheprior6weeks,aberrantdiurnal criteria wereknowndiseaseintheHPA axis, care centres,localnewspapers,andsocialmedia.Exclusion advertisements atthehospital,university, health primary years of age was recruited from Umeå, Sweden, through ( 2.5th and97.5thpercentiles with95%confidencelimits least 153referenceindividualstobeablecalculatethe we aimed to prospectively include at reference intervals Institute (CLSI)approvedguidelinesforestablishing Standards In accordance with the Clinical and Laboratory diagnosis Salivary cortisoneinCushing > 19 38 ). Thereferencegroupofmenandwomenover18 The presentstudyaimedtoestablishrobustreference Of the175referencesubjectsrecruited,17 ° C), and severe oral problems. After providing written n

= 5), andpregnancy ( n

= 4), severe oral problems ( Downloaded fromBioscientifica.com at09/25/202110:23:41PM 20 n ) Frhroe three Furthermore, =6). ). Participatingwomen n 182 ) msig blood missing =1), :6 ≥ n 138 nmol/L ) serious =1), 570 via freeaccess European Journal of Endocrinology BMI, body mass index; CS, Cushing’s syndrome; ISI, insomnia severity index. No Yes Hypertension None Mild Oral problems ISI score Snus users No tobaccouse Smokers Tobacco use Age No Yes Diabetes BMI 61-70 y 51-60 y 41-50 y 31-40 y 20–30 y Age All Study cohort Table 1 at UmeåUniversity(Dnr2015/08-31). in of thepatientswithCS( confirmed CSwereincluded inthestudy. Characteristics these werethereforeexcluded.Thus,onlypatientswith patients inthisgroupCScouldnotbeconfirmedand according toastandardizedquestionnaire.Intwoofthe of CSwerereportedbytheparticipatingendocrinologists, clinical andbiochemicalcharacteristics,typeseverity subjects wereincluded.ConfirmedCSdiagnosisincluding Sweden, duringthesameperiodoftimethatreference endocrine departmentsoffiveuniversityhospitals in of agewithastrongsuspicionCSwereincluded at cortisolandcortisone,24patients salivary ( Baseline characteristicsoftheeligiblereferencegroup 13 had plasma cortisol levels of 50–137 nmol/L after DST. ( n 21 < ≥ < ≥ < ≥ ≥ Clinical Study

= 15 15 70 y 70 y 30 kg/m 71 y Table 2 30 kg/m ). Oftheremaining155includedreferencesubjects, 155) arepresentedin To testthesensitivityandspecificityforCSusing This studywasapprovedbythe localEthicsCommittee Baseline characteristicsofthereferencepopulation. 2 . 2 Women 91 77 14 84 90 77 82 88 77 14 14 13 22 11 24 7 1 9 5 9 3 7

n Table 1

= Men 54 10 64 0 58 6 11 51 2 58 6 61 3 58 6 6 5 16 12 11 14 64 22) inthestudyarepresented

. N Bäcklundandothers population 131 (85) 148 (95) 148 (95) 128 (83) 140 (90) 149 (96) 135 (87) 155 (100) Reference 24 (15) 20 (13) 15 (10) 20 (13) 13 (8) 19 (12) 29 (19) 34 (22) 22 (14) 38 (25) 7 (5) 7 (5) 7 (5) 6 (4)

(%) > with CS 18 years 16 (73) 18 (82) 15 (68) 13 (59) 22 (100) 14 (64) 13 (59) 13 (59) Patients 2 (9) 4 (18) 7 (32) 9 (41) 4 (18) 3 (14) 5 (23) 8 (36) 1 (5) 1 (5) 8 (36) 9 (41) 6 (27) 4 (18)

(%) Missing data Severe Moderate Mild Severity, Missing data Adrenal adenoma Ectopic ACTH Cushing’s disease Aetiology, Age, y;mean(range) Women, Characteristics Cushing’s syndrome. Table 2 h, 23:00handthenextmorningat08:00afterDST. All samples atoneoccasion,fortwoconsecutivedays08:00 March 2015toApril2016. Eachindividualcollected Saliva samples were collected at participants’ homes from Saliva collection samples wereseparatedwith ultra-HPLC(UHPLC)on as describedpreviously(second setupin( The cortisolinsalivawas quantified withLC-MS/MS, Biochemical analysismethods the samebatchassamplesfromreferencesubjects. CS atotherhospitalsweresentbymailandanalysed in until analysis. Saliva samples collected from patients with from eachtimepointwerepooledandstoredat centrifuged (5minat2826 were frozenat was drawnforplasmacortisolanalysis.AllSalivette collected at08:00h( and onthefollowingday, duplicatesalivasampleswere (Abcur AB,Helsingborg,Sweden)wastakenat23:00h one at22:00( population alsoprovidedonesalivasampleat20:00hand h ( participant providedduplicatesamples,at08:00and23:00 for 1½ min, to allow passive saliva absorption. Each Cortisol swabfor1minorbyplacingitunderthetongue Nümbrect, Germany)eitherbychewingontheSalivette was collectedusingSalivette mouth withwaterat10minpriortosampling.Saliva teeth for30minpriortosampling;andrinseoutthe h before sampling; to avoid eating, drinking, and brushing avoid smokingandsnus(moist,smokelesstobacco)for1 activity onthedayofcollectionandbefore;to participants wereinstructedtoavoidstrenuousphysical diagnosis Salivary cortisoneinCushing ± 15 min).Onthesame sampling day, half the reference n n Demographic characteristicsofpatientswith (%) (%) n (%) ± − 15 min).A1-mgdexamethasonetablet 20 ° C foratleast24h,thenthawedand ± 15 min)andavenousbloodsample Downloaded fromBioscientifica.com at09/25/202110:23:41PM g ). Next,theduplicatesamples ® Cortisoltubes(Sarstedt, https://eje.bioscientifica.com 182 Patients :6 12 (55) 55 (22–82) 17 (77) 1 (5) 5 (23) 8 (36) 8 (36) 1 (5) 4 (18) 5 (23) 22 ( n )). Briefly, = 22) ® − tubes 571 80 ° via freeaccess C ®

European Journal of Endocrinology https://eje.bioscientifica.com recommendations oftheCLSI ( were calculated according to the Reference intervals reference samples Defining thereferencepopulation and previously ( 3.3 nmol/LthemorningafterDST, asdescribed university hospital,withalowerlimitofnormal of dexamethasone wasanalysedwithLC-MS/MSatBergen the methodin use atthetreatinghospital.Plasma cortisol levels in patients with CS were analysed with hospital. The CV was II reagentsonCobase602analyseratUmeåUniversity analysed consecutivelywithRocheElecsysCortisolIand were 0.25nmol/Land0.20nmol/L,respectively. prepared samples.TheLLoQsforcortisolandcortisone ± noise ratiowasgreaterthan10:1;accuracywithin defined asthelowestconcentrationwheresignal-to- 110 nmol/L.Thelowerlimitofquantification(LLoQ)was CVs for cortisone were 7% at both control levels, 4 and at controllevelsof1.4and50nmol/L,respectively. The coefficients ofvariation(CVs)forcortisolwere6and7% (C2755, Sigma-Aldrich) in methanol. The inter-assay (H4001, Sigma-Aldrich)and2.77mmol/Lcortisone from stocksolutionscontaining2.76mmol/Lcortisol vol) PBS, pH 7.4,at 0, 1,5, 20, 50, 150, and 300 nmol/L, losses. Calibrationstandardswerepreparedin0.1%(wt/ used asinternalstandardstocompensateforrecovery 2218, CambridgeIsotopeLaboratories,Larodan)were Sigma-Aldrich) andcortisol-9,11,12,12-d Deuterated cortisone-2,4,6,12,12-d 100 and104%at5nmol/Lrespectively. 5 nmol/Land50respectively, andforcortisone (361.2 the cellexitpotentialswere15V(361.2 were 32V(361.2 potential forcortisonewas75V, thecollisionenergies and confirmedatm/z361.2 it wasquantifiedbyatransitionatm/z361.2 analysed inthesamerun.Cortisoneeluted at 3.47 min; 20 or bythetwoendogenouscortisolisomers,20 by samplesfrompatientstreatedwithprednisolone This methodwaspreviouslyshowntobeunaffected with electrosprayionizationinthepositiveionmode. with a SCIEX QTRAP 5500 LC-MS/MS system, operated a KinetexBiphenyl column. Analysiswasperformed 20%; andimprecisionwaslessthanCV20%forten Clinical Study β -dihydrocortisone. Cortisoneandcortisolwere Plasma cortisol levels in reference individuals were >

121.1). Recovery forcortisolwas78and79%at 121.1). Recovery 24 ). >

163.2) and38V(361.2 < 6% at 300 nmol/L ( > 19

121.1. Thedeclustering N Bäcklundandothers ). >

163.2) and13V > 2.) and 121.1), 23 7 (705586, ). Plasma 4 (DLM- > α 163.2 - and

conducted byrepeatedlydichotomizingthereference population ( points werecomparedbetweensubgroupsofthereference cortisolandcortisonelevelsatseparatetime Salivary Subgroup comparisons implemented forsamplescollectedat20:00and22:00h. outlier ( total rangeofallvalues,thathighvaluewasconsideredan highest valueandthesecondwas Dixon method; that is, when the difference between the excluded fromthereferencesamples,accordingto exogenous hydrocortisone.Then,extremeoutlierswere samples), forsuspicionofcontaminationwithbloodor above the 97.5th percentile were excluded ( criteria. Individual samples with a cortisol:cortisone ratio steps ofexclusion,inadditiontothepredefinedexclusion time points,includingchanges inthecortisol:cortisone samples collectedfromthe sameindividualatdifferent Wilcoxon signed-rank testwasusedwhencomparing ratios betweenthereference groupandtheCSgroup.The which was also used when comparing cortisol:cortisone population werecomparedwiththeMann–Whitney the bootstrapmethod.Subgroupswithinreference and the95%CIsofthesepercentiles werecalculatedwith were calculated with the 2.5th and 97.5th percentiles, only non-parametricstatisticswasused.Referenceranges Our datadidnotfollowaGaussiandistribution;therefore, Statistical analysis were excludedfromtheseanalyses. was notconfidentlyestablished;therefore,thesesubjects comparison was made. In two women, menopausal status hormonal replacementtherapy(HRT)andthereforeno ( postmenopausal useoflocaloestrogens,ornooestrogens intrauterine device,ornoexogenousoestrogens;and premenopausal use ofoestrogencontaining OC, hormonal and postmenopausalfemalereferencesubjects; several subgroupsofwomen,includingpremenopausal (ISI scoreunderor for hypertension (Y/N), andsignificant sleepdisturbances (Y/N), diabetescurrentpharmacologicaltreatment smoking orusingsnus),self-reportedmildoralproblems ≥ population, basedongender(male/female),age(underor diagnosis Salivary cortisoneinCushing Table 4 70 years), BMI (under or To evaluatetheeffectofoestrogenstatus,wecompared Defining theeligiblereferencesamplesincludedtwo n ). Onlyonepostmenopausalwomanusedoral

= 6 of 455 samples) ( Table 3 ≥ 15). ). Thus,subgroupanalyseswere Downloaded fromBioscientifica.com at09/25/202110:23:41PM ≥ 30 kg/m 19 ). The same procedure was 2 ), tobacco use (Y/N to 182 :6 n 0 f 465 of =10 > 1/3 ofthe U 572 test, via freeaccess European Journal of Endocrinology OC, estrogencontainingoralcontraceptives; IUD,hormonalintrauterinedevice. * Pairs ofgroupsthatdid/didnotuseoestrogen werecomparedwiththeMann–Whitney Postmenopausal + Postmenopausal Premenopausal + IUD Premenopausal Premenopausal + OC Postmenopausal (all) Premenopausal (all) Subgroup ofwomen reference population. Table 4 Group subgroups withinthereferencepopulation. Table 3 Diabetes BMI Male Female All Age No Yes Hypertension No Yes Tobacco use Oral problems ISI score Snus users No tobacco Smokers BMI, bodymassindex;ISI,InsomniaSeverityIndex. * that didnotusetobacco(notobacco). Pairs ofgroupswerecomparedwiththeMann–Whitney No Mild P P < ≥ < ≥ < ≥

Clinical Study local oestrogens no oestrogens no oestrogens < < 30 30 70 70 15 15 0.05. 0.05, ** Oestrogen effectsonsalivarycortisolandcortisone,measuredat08:00h,23:00afterDSTinwomenthe Comparison ofthesalivarycortisolandcortisonevaluesmeasuredat08:00h,23:00afterDST,indifferent P

< 0.01.

155 135 149 140 131 128 148 148

20 64 91 24 15 20 n 6 7 7 7

17.0** (9.0–23.6) 20 45 30 59 n 9 9 5 10.4 (5.4–13.5) 9.1 (5.8–12.8) 8.3 (5.1–13.2) 8.4 (5.2–13.0) 8.7 (5.3–13.2) 7.6 (5.0–10.9) 8.3 (5.2–13.0) 8.9 (4.9–13.8) 8.3 (5.2–13.1) 9.6 (7.9–10.9) 8.2 (5.2–11.5) 8.3 (5.2–13.0) 8.7 (5.9–15.0) 8.4 (5.2–13.1) 8.4 (5.2–12.8) 8.4 (5.2–13.1) 8.4 (5.8–10.2) 08:00 h 14.0* (8.3–16.4)

10.2 (8.0–14.2) 8.3 (5.3–13.0) 8.4 (5.8–14.6) 8.2 (4.7–16.0) 8.2 (4.9–11.7) 7.7 (4.8–15.1) 08:00 h N Bäcklundandothers Median salivarycortisol, Median salivarycortisol, (25th–75th percentile) 1.5** (0.85–2.2) 0.60 (0.35–1.3) 0.73 (0.40–1.3) 0.68 (0.40–1.3) 0.69 (0.40–1.3) 0.61 (0.38–1.3) 0.65 (0.39–1.1) 0.67 (0.39–1.1) 0.91 (0.40–1.4) 0.66 (0.39–1.1) 0.65 (0.39–1.1) 0.71 (0.39–1.3) 0.60 (0.48–0.68) 0.68 (0.39–1.3) 0.67 (0.43–0.95) 1.7* (1.0–3.4) 1.2* (0.54–1.8) (25th–75th percentile) 1.6 (0.43–1.7) U 0.85 (0.55–1.1) 0.52 (0.34–1.5) 0.67 (0.43–1.4) 0.73 (0.39–1.0) 0.95 (0.71–4.8) 0.72 (0.38–1.4) 0.73 (0.40–1.2)

23:00 h test.Smokersandsnus(moistsmokelesstobacco)userswerebothcomparedtoindividuals 23:00 h nmol/L 0.17 (0.11–0.27) 0.19 (0.15–0.27) 0.15 (0.11–0.25) 0.19 (0.12–0.27) 0.17 (0.11–0.26) 0.16 (0.11–0.27) 0.21 (0.16–0.27) 0.17 (0.11–0.26) 0.26 (0.17–0.54) 0.17 (0.11–0.25) 0.25 (0.16–0.35) 0.17 (0.11–0.27) 0.23 (0.09–0.37) 0.17 (0.11–0.27) 0.18 (0.10–0.25) 0.18 (0.13–0.23) 0.17 (0.11–0.27) 0.19 (0.08–0.26) nmol/L 0.26 (0.14–0.45) 0.21 (0.16–0.28) 0.19 (0.12–0.33) 0.15 (0.11–0.27) 0.23 (0.19–0.49) 0.21 (0.16–0.28) 0.18 (0.11–0.29)

diagnosis Salivary cortisoneinCushing DST

DST

U test. 40.1* (32.2–57.9) 34.8* (25.6–42.2) 36 .3(26.3–39.8) 28.7 (22.8–37.6) 28.9 (22.0–36.4) 28.9 (22.3–36.7) 28.7 (22.2–37.1) 31.1 (25.4–34.4) 28.5 (22.7–36.7) 29.8 (19.0–37.3) 28.7 (22.5–36.6) 28.7 (22.2–37.6) 27.9 (22.1–35.1) 29.1 (22.2–37.6) 26.6 (22.3–34.1) 29.0 (22.8–36.9) 22.2 (20.0–30.9) 28.6 (22.2–36.5) 31.6 (26.6–39.2) 29.4 (21.1–33.7) 29.1 (20.0–36.2) 28.7 (21.8–36.8) 34.0 (23.0–35.6) 27.2 (21.2–34.7) 30.0 (22.5–36.9) 08:00 h 08:00 h Median salivarycortisone, Median salivarycortisone,

Downloaded fromBioscientifica.com at09/25/202110:23:41PM (25th–75th percentile) (25th–75th percentile) 6.9* (5.4–9.7) 5.4 (3.9–7.0) 3.5 (2.4–6.3) 4.8 (3.9–6.2) 4.3 (3.2–7.5) 5.6 (5.0–12.5) 4.0 (2.9–6.3) 4.7 (3.4–6.4) 4.3 (3.0–7.0) 4.1 (2.6–6.0) 3.6 (2.9–8.0) 4.5 (3.2–6.3) 4.3 (3.0–7.0) 4.3 (3.0–7.0) 5.7 (2.9–7.3) 4.3 (3.0–6.7) 8.5 (4.7–12.3) 4.0 (3.0–6.4) 4.3 (3.0–6.9) 6.3 (3.4–10.1) 4.3 (3.0–7.0) 4.8 (3.0–7.9) 4.3 (2.7–8.1) 4.3 (3.0–7.1) 4.8 (3.7–5.8) https://eje.bioscientifica.com

23:00 h 23:00 h 182 :6 nmol/L nmol/L 1.4 (1.1–1.6) 1.1 (1.0–1.5) 1.1 (0.88–1.4) 1.5 (1.1–1.9) 1.7 (1.2–2.3) 1.2 (1.0–1.6) 1.5 (1.1–1.9) 1.6* (1.4–2.5) 1.3 (1.0–1.7) 1.4 (1.0–1.7) 1.2 (1.0–1.6) 1.3 (1.1–1.8) 1.3 (1.0–1.7) 1.2 (1.0–1.7) 1.4 (1.2–1.7) 1.3 (1.0–1.7) 1.6 (1.2–2.6) 1.2 (1.0–1.7) 1.3 (1.0–1.7) 1.4 (1.0–2.0) 1.3 (1.0–1.7) 1.4 (1.0–2.0) 1.2 (1.0–1.9) 1.3 (1.0–1.7) 1.2 (1.0–1.4)

DST DST

573 via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com The arrowindicatesthe97.5th percentile. Distribution of salivary cortisol:cortisone ratios in 720 samples. Figure 1 contaminated withbloodorexogenoushydrocortisone, cortisol:cortisone ratios value was0.81( of 1.06,thenextclosestratioto97.5thpercentile percentile was1.07.Apart fromonesamplewitharatio median cortisol:cortisone ratio was 0.17 and the 97.5th or cortisonebelowtheLLoQratiowassetto0.The the patientswithCS( reference group ( samples,fromalltimepoints,boththe 720 salivary cortisol:cortisoneratiowascalculatedfrom The salivary Salivary cortisol:cortisoneratio Results were consideredsignificant. IBM SPSSStatistics,version25(IBMcorp.). of thesetests.Allothercalculationswereperformedwith cortisoland cortisone after DST and combinations salivary cortisoneat23:00h, diagnostic tests,includingsalivary standard when comparingtestaccuracy for novel with the bootstrap method. LNSC was used as reference and tocalculatethe95%CIsforsensitivityspecificity (AUCs) was usedtocompareareasundertheROCcurves software package,Rv3.5.2(RCoreTeam, Vienna, Austria), analyses wereperformedforeachtest,andthestatistical reference group).Receiver-operatingcharacteristic(ROC) upper referencelimit(i.e.,the97.5thpercentile forthe identifying patients with CS was performed using the ANOVA. Sensitivityandspecificitytestsforcorrectly were evaluated with the Kruskal–Wallis non-parametric ratio overtime.Seasonal differences (3-month periods) Clinical Study Fig. 1 n

= 155), pregnant women ( ). Therefore,allsalivasampleswith n ≥

= 1.0 wereconsideredpotentially 22). Forsampleswithcortisol N Bäcklundandothers P values n ) and =6) < 0.05 Reference intervals vs 0.29). not for the unsuppressed samples taken at 08:00 h (0.32 0.14) andafterDST(0.28vs0.12; reference populationforsamplestakenat23:00h(0.28vs significantly higherintheCushingpopulationthan cortisol:cortisoneratioswere for both).Thesalivary higher at08:00hthan23:00andafterDST( cortisol:cortisoneratiowassignificantly The salivary 0.36), 0.14(0.11–0.21),and0.12(0.09–0.17),respectively. percentiles) cortisol:cortisoneratiosof0.29(0.23– salivary 08:00 h,23:00andafterDSThadmedian(25th–75th after exclusionofthesesamples).Samplescollectedat below the97.5thpercentile ateachtimepoint(calculated cortisol:cortisone ratios samples). Notably, allreferencesamplesexcludeddueto and excludedfromfurtheranalyses( sensitivity andspecificity( discriminate CSfromthereference populationwithhigh were included( inhaled, ornasalglucocorticoids andextremeoutliers were excluded, whereas referenceindividuals using local, analyses, onlysampleswithcortisol:cortisoneratios diagnostic situationascloselypossibleinthese ascut-offvalues( intervals with CS wastestedusing the upper limits of the reference cortisone at23:00handafterDST, toidentifypatients cortisoland specificity oftheindextests,thatissalivary to thereferencepopulation( and cortisonelevelsat23:00hafterDST, compared cortisol Patients with CS had considerably higher salivary from thereferencepopulation Discriminating patientswithCushing'ssyndrome missing bloodsamples, complete the DST (pregnant women and subjects with 164 referencesubjects,excludingonlythosethatdidnot was 11–134nmol/L(median25nmol/L),calculatedfrom and 0.59–3.5nmol/LafterDST( 14.4–57.5 nmol/Lat08:00h,1.5–13.523:00 cortisonewere calculated for salivary reference intervals 23:00, and were 2.7–22.9nmol/Lat08:00h, (2.5th–97.5thpercentile) cortisol intervals forsalivary For theeligiblereferencegroup( diagnosis Salivary cortisoneinCushing We cortisol and cortisone could found that salivary forplasmacortisol After DSTthereferenceinterval < 0.25–0.79 nmol/LafterDST( n =164). n ≥ Downloaded fromBioscientifica.com at09/25/202110:23:41PM =11). 1.0 had salivary cortisonelevels 1.0 hadsalivary Fig. 3 Table 5 Fig. 3 and Fig. 2 ). To mimictheclinical P n

). Thesensitivityand < 5) te reference the =155), < Table 5 182

). 0.001 forboth),but 0.25–3.6 nmol/Lat :6 n ). Notably, the 0 f 720 of =20 Fig. 2 P

< ). The 0.001 574 ≥ 1.0, via freeaccess European Journal of Endocrinology levels (i.e. cortisol and cortisone accuracy of the sum of salivary glucocorticoids, wealsotestedthediagnostic on salivary DST withplasmacortisol. from thediagnosticaccuracyofLNSCorconventional measured after the DST were not significantly different cortisolandcortisonelevels AUCs)ofthesalivary curve the endofthisarticle).Thediagnosticaccuracies(ROC Fig. 1,seesectionon cortisol at23:00h)(AUC:0.976; 0.985) was significantly higher thanthat ofLNSC(salivary cortisone at23:00h(AUC: AUCforsalivary ROC curve suppression test. MS-method ( was belowthelowerlimitofquantificationforLC-MS/ salivary cortisolat23:00handafterDST,the2.5thpercentile indicate themeanvalue(95%CI)ateachboundary.For of eachinterval.Greyshadedboxesandthevaluesbelow Dashed linesmarkthe2.5thand97.5thpercentileboundaries time points.AllvaluesontheX-axesareexpressedinnmol/L. salivary cortisone(B,DandF),measuredatthreedifferent Reference intervalsforsalivarycortisol(A,CandE) Figure 2 Clinical Study To evaluatenovel approachesinCSdiagnosticsbased ∑ < ), measuredeitherat23:00h oraftertheDST. 0.25 nmol/L).DST,overnightdexamethasone supplementary materials supplementary P N Bäcklundandothers 0.029) (Supplementary (Supplementary =0.029) givenat in 153referenceindividualsthemorningof Plasma dexamethasoneconcentrationsweremeasured with discrepantresults Plasma dexamethasoneandreferencesubjects ( cortisonemeasurementsat23:00handafterDST salivary The sameresultswerefoundforthecombinationof cortisolvaluesorthez-scoresforeachtimepoint. salivary These results were similar, whether we used the absolute cortisone. over LNSC,butnotthelate-nightsalivary combined testalsoshowedsuperiordiagnosticaccuracy 23:00 cortisolafterDST(i.e. cortisol at23:00handthesalivary We alsoevaluatedwhetheracombinationofsalivary cortisonealone. diagnostic valuethanlate-nightsalivary than theLNSC( The formershowed significantly better diagnosticvalue < (interquartile range(IQR)):31 ( higher inthereferencesubjects the DST. PlasmacortisolafterDSTwas alsosignificantly cortisone at 23:00 h and after significantly elevated salivary ≥ in individualswithhypertension,diabetes,andage cortisol at23:00hwasslightly, butsignificantly elevated population arepresentedin between differentsubgroupswithinthereference cortisolandcortisone levels Comparisons ofsalivary Subgroups inthereferencepopulation excluding theseindividuals). (established after levels within the reference intervals cortisolandcortisone subjects hadlate-nightsalivary concentrations Of thesethreesubjects,twohadplasmadexamethasone excluded afterrepeatedfollow-upandbiochemicaltests. clinical manifestationsofhypercortisolism, andCSwas from theeligiblereferencepopulation.Allthreelackedany cortisol cortisol dexamethasone cortisol 50–137nmol/L.Ofthe13subjectswithplasma dexamethasone ( sufficient tosuppressplasmacortisolbelow50nmol/L plasma dexamethasoneconcentrations DST. Aprevious studyusingthismethodindicatedthat diagnosis Salivary cortisoneinCushing Table 5 25 70, median:24( 70 years.Referencesubjects ). Ofthe140referenceindividualswithplasma + DST) mightincreasethediagnosticaccuracy. This ). > ≥ 50 nmol/L. Three reference subjects had plasma 138 nmol/LaftertheDST; thesewereexcluded < ≥ < 3.3 nmol/L.Notably, allthesethree 18 P 3.3 nmol/L, nine (6.4%)had plasma 3.3 nmol/L, four (31%) had plasma

= – 0.049), butneithershowedbetter 31 ) nmol/L( Downloaded fromBioscientifica.com at09/25/202110:23:41PM ≥ Tables 3 27 https://eje.bioscientifica.com 70 yearsofagealsohad – P 52 .0) Soes had Smokers =0.005). 182 ≥ ) nmol/Lvssubjects 70 years,median ≥ and :6 3.3 nmol/Lwere 4 . Salivary . Salivary 575 via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com had higher median salivary cortisol and cortisone levels had higher median salivary nmol/L ( exogenousoestrogen ( ( higher plasmacortisollevelsafterDST, median(IQR):50 used oestrogencontainingOC( premenopausal women.Premenopausalwomenthat cortisol orcortisonebetweenpostmenopausaland not usetobacco(median:0.28, (median: 0.41)at08:00h,comparedtosubjectsthatdid also hadasignificantly highercortisol:cortisone ratio cortisolat 08:00h.Smokers significantly elevatedsalivary in themorning at 08:00, and subjects that used snus had cortisolandcortisonelevels significantly elevatedsalivary evaluated ateachtimepoint.DST,overnightdexamethasonesuppressiontest. the reference population (blue) and patients with CS (red). Arrows indicate the 97.5th percentile value of the reference population, Comparison ofsalivarycortisol(AandC)cortisone(BD)levels,measuredat23:00hB)afterDST(C D)in Figure 3 39 Clinical Study – There were no significant differences in salivary There werenosignificantdifferencesinsalivary 77 ) nmol/L,thanpremenopausal womenwithout P

= 0.007). Premenopausalwomen usingOCalso n

= 45); median(IQR):26( P =0.035). N Bäcklundandothers n ) a significantly had =5) 20 – 35 ) not performedinthepregnant women. calculated frommenandnon-pregnant women.DSTwas (15.3 and18.3nmol/L)based ontheupperlimitofnormal cortisone women hadslightlyelevated late-nightsalivary premenopausal women.However, twoofthepregnant was notsignificantlydifferentfromthenon-pregnant 2.4) nmol/Landcortisonewas5.7(4.4–13.2).This cortisolat23:00hwas 1.4(0.64– median (IQR)salivary levels after DST ( levels ( cortisol orcortisone oestrogens hadnoeffectuponsalivary morning (08:00h)cortisollevels( oestrogen, butthedifferencewasonlysignificantfor than premenopausalwomenthatdidnotuseexogenous Among thepostmenopausalwomen( diagnosis Salivary cortisoneinCushing Table 4 ), nordidithaveeffectonplasmacortisol P

= 0.281). In the pregnant women ( Downloaded fromBioscientifica.com at09/25/202110:23:41PM 182 P n =0.045;

= :6 30), useoflocal Table 4 n 576 =6), via freeaccess ). European Journal of Endocrinology No significantdifferencewasfound amongthedifferentseasons(Kruskal–WallisANOVA). Winter (Dec-Feb) Season season. Table 6 Autumn (Sep-Nov) Summer (Jun-Aug) Spring (Mar-May) suppression test;LNSC,latenightsalivarycortisol;P-cortisol,plasmaROC,receiver-operatingcharacteristic. ∑ *23:00 + DST wascalculatedbasedoncombinedZ-scoresfor23:00andDSTeachmeasurand. Salivary cortisone Salivary cortisol 23:00 + DST P-cortisol Salivary cortisone Salivary cortisol DST Salivary cortisone LNSC 23:00 h Diagnostic test Table 5 P nmol/L; those collectedat23:00h(mediancortisol:0.56vs0.60 was foundbetweensamplescollectedat22:00hand vs 3.53nmol/L; cortisol 0.72vs0.60nmol/L; at 20:00hcomparedtothosecollected23:00(median levels werebothsignificantlyhigherinsamplescollected 23:00 h.Within cortisolandcortisone thisgroupsalivary ( sampling timepoints.Halfofthereferencepopulation Finally, weevaluatedtheeffectsofdifferentevening Effect ofeveningtimepoint season ( and autumn(Sep–Nov)showednosignificanteffectsof winter (Dec–Feb),spring(Mar–May),summer(Jun–Aug), h, 23:00 h, and after DST in samples collected during cortisolandcortisonelevelsat08:00 Comparing salivary Seasonal effects n =0.603; , thesumofcortisolandcortisoneatindicatedtimepoint;AUC,areaunde Clinical Study ∑ ∑

= 78) collectedsalivasamplesat20:00h,22:00hand Table 6 P Seasonal differencesinsalivarycortisolandcortisonelevels, measuredat08:00h,23:00andafterDSTduringeach Diagnostic testaccuracyforidentifyingCSwithsalivarycortisoland/orcortisone.

Fig. 4 = * 0.087; mediancortisone:3.76vs3.53nmol/L; ). ). Themedianreported‘normalbedtime’ P 39 35 51 30 n < 0.001). Nosignificantdifference 97.5 percentile 7.9 (4.8–15.2) 8.2 (4.8–11.5) 9.5 (5.9–14.6) 8.8 (5.8–12.8) (nmol/L) 0.79 08:00 h 16.4 13.5 3.6 134 3.9 3.5 P N Bäcklundandothers .1; otsn 5.70 cortisone =0.014; Median salivarycortisol,

(25th–75th percentile) 0.70 (0.42–1.3) 0.68 (0.40–1.3) 0.85 (0.33–1.4) 0.56 (0.40–1.1) Sensitivity, 100 (83–100) 100 (83–100) 100 (84–100) 100 (84–100) 23:00 h 95 (75–100) 95 (75–100) 90 (70–99) (95 %CI) %

nmol/L 0.20 (0.13–0.31) 0.16 (0.10–0.26) 0.16 (0.12–0.22) 0.21 (0.10–0.37) Specificity,

22:37–23:30 h), only twonormally going tobed before for thispartofthereferencepopulationwas23:00h(IQR cortisol to cortisone in the salivary gland. cortisol tocortisoneinthesalivary by sample contamination or the variable conversion of cortisol, caused the putativeriskofafalsehighsalivary and is hamperedbythelackofsolidreferenceintervals sample collectionandhighaccuracy. Thetesthowever dexamethasone metabolism. The LNSC benefits from easy example, variations in plasmaCBGconcentrations and often yieldsfalsehighorgrey-zonevalues,dueto,for impairment ( and isdifficulttoevaluateinpatientswithmoderaterenal collections; havelowsensitivityfordetectingmildCS; collection forthepatient;areoftenincomplete24-h accuracy. The24-hUFCrequirestrenuoussampling simple biochemicalscreeningtestswithhighdiagnostic Improving theprocedurefordiagnosingCSrequires Discussion 22:00 handnonebefore21:00h. DST 98 (94–100) 95 (90–98) 94 (89–97) 96 (92–99) 95 (90–98) 95 (90–98) 96 (91–98) diagnosis Salivary cortisoneinCushing r thecurve;CS,Cushing’ssyndrome;DST,overnightdexamethasone (95 %CI) %

26.1 (20.9–34.5) 29.8 (23.4–37.1) 31.7 (25.5–39.2) 29.4 (21.4–37.9) 26 , 08:00 h 0.993 (0.985–1.000) 0.985 (0.970–1.000) 0.997 (0.992–1.000) 0.993 (0.985–1.000) 0.993 (0.985–1.000) 0.988 (0.971–1.000) 0.983 (0.966–1.000) 0.985 (0.970–1.000) 0.976 (0.955–0.996) 27 ). TheclassicDSTwithplasmacortisol Median salivarycortisone, ROC-curve AUC (95 %CI) Downloaded fromBioscientifica.com at09/25/202110:23:41PM (25th–75th percentile) 4.4 (3.2–6.4) 5.1 (2.6–8.2) 4.4 (3.0–7.7) 3.6 (3.1–6.0) https://eje.bioscientifica.com 23:00 h

182 :6 ROC-curve vsLNSC nmol/L 1.4 (1.0–1.9) 1.2 (0.91–1.8) 1.4 (1.1–1.7) 1.2 (0.87–1.5) P valuefor 0.023 0.041 0.050 0.109 0.097 0.315 0.049 0.038

– DST

577 via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com primary measurand. primary cortisone should be the this result suggests that salivary at 23:00wassignificantly higherthanthatofcortisol; cortisone upper referencelimits.The accuracy ofsalivary identifying patientswithCSusingthenewlyestablished highsensitivityandspecificity for after DSTallhavevery cortisoland cortisone cortisone at23:00handsalivary and thelessestablishedbiomarkers/tests,thatissalivary showed thatboththeestablishedbiomarkerforCS,LNSC, patients withCSthereferencepopulationwe and afterDST, measuredwithLC-MS/MS.Bycomparing cortisone at 23:00 h cortisol and salivary for salivary * percentiles andwhiskersarethe2.5th97.5thpercentiles, cortisol andcortisonelevels.Boxesrepresentthe25th–75th Effects ofdifferenteveningsamplingtimepointsonsalivary Figure 4 P Clinical Study

< 0.05 withtheWilcoxonsigned-ranktest. In thisstudy, weestablishedrobustreference intervals N Bäcklundandothers found a higher salivary cortisol:cortisoneratioinsamples found ahighersalivary the salivasample( ratings ormeasurementsofhemoglobintransferrinin be addressed by other methods, including discoloration by riftsintheoralmucosa).Thelattermayhoweveralso increased bybloodcontaminationofthesalivasample(i.e. contamination with topical hydrocortisone but is also useful to detect sample cortisol:cortisone ratio is very acid orgrapefruitjuice)( intrinsic enzymevariationsorinhibitionbyglycyrrhizic variation in11 ratios above1,due to potentialsample contamination or supported theexclusionofsampleswithcortisol:cortisone ratio of0.18anda97.5thpercentile of1.07.Thesefindings cortisol:cortisone cortisone revealedamediansalivary cortisoland Our simultaneousquantificationsofsalivary and theabilitytoanalysesimilarsteroidssimultaneously. common, duetoadvantagesincludinghighspecificity salivary cortisol,consistentwithpreviousstudies( salivary to beslightly, butsignificantlybetterthanlate-night cortisone patients withCSwefoundlate-nightsalivary cortisone showedhighdiagnosticaccuracyforidentifying ( LC-MS/MS, werecomparabletothosepreviouslyreported cortisolandcortisonelevels,measuredwith night salivary gland11 salivary 08:00 handfrompatientswithCS),indicatingthatthe with higherplasmacortisollevels(i.e.samplestakenat reflect the free plasma cortisol level at each time point. cortisone at23:00h(orafter DST)couldpotentiallybetter cortisolandsalivary combined tests.Thesumof salivary previously ( cortisol,assuggested gland),comparedtosalivary salivary to sample contamination or low 11 higher stability and a lower risk of false results (e.g. due cortisone forCSscreeninganddiagnosismightprovide together, these findingsindicatethatanalysingsalivary Takenvalues within the calculated reference intervals. cortisol:cortisone ratios( reference subjectsthatwereexcludedduetohighsalivary plasma cortisol. Furthermore, it should be noted that all and bothwerenoninferiortotheconventionalDSTwith cortisolandcortisone, diagnostic accuracyofsalivary cortisol andcortisone.AlsoafterDSTwefoundhigh the DST, forsalivary after establishingreferenceintervals 25 diagnosis Salivary cortisoneinCushing 8 , , 35 35 quantificationwithLC-MS/MSisincreasingly Our stringently acquired reference intervals forlate- Our stringentlyacquiredreferenceintervals We alsoevaluated the diagnosticaccuracyofdifferent , ). We alsoexploredtheuseofsalivasamplesafter 36 ). While both late-night salivary cortisol and ). While both late-night salivary 16 , β 35 -HSD2 activity in the salivary gland(e.g., -HSD2 activityinthesalivary β ). -HSD2 wassaturated( 33 ). Consistentwithotherstudies,we 28 Downloaded fromBioscientifica.com at09/25/202110:23:41PM ≥ , 29 1.0) had salivary cortisone 1.0) hadsalivary , 30 , 31 β -HSD2 activity in the 182 , 32 6 :6 , ). Elevatedsaliva 9 , 34 ). 3 , 8 578 , 15 via freeaccess , European Journal of Endocrinology salivary cortisone after DST,salivary in line with previously shown cortisol and cortisonelevelsat23:00h salivary reference subjectsover70years hadsignificantlyhigher subjects with hypertension and diabetes. Furthermore, cortisollevelsat23:00in significantly elevatedsalivary 49 in thosegroups( diseases. Thus,thediagnosisofCSismoredifficult enousoestrogen use,sleepdisturbances,andcommon tobacco use,exog­ increase withaging,overweight, hypercortisolaemic states. remain constant throughout the year, when evaluating DST. can Thisfindingindicatesthatthereferenceintervals cortisone levels,measuredateither23:00horafter cortisolor significant seasonaldifferencesinsalivary no seasonally affect cortisol secretion, we observed substantial variationsinthesefactorssuggestedto to considerableseasonaltemperaturevariations.Despite h of daylight in June and 4 h in December, in addition (northern Sweden),whichexperiencesapproximately21 reference samplesoveraone-yearperiodinUmeå a seasonalvariation( known diurnalvariation,cortisollevelsmightalsohave night samplesusingthesereferenceintervals. but thatsamplingat20:00hisnotrepresentativeaslate- cortisol andcortisonemaybecollectedat22.00horlater samplingfor suggests that unstressed late-night salivary collected at20:00hhadsignificantlyhigherlevels.This cortisone collectedat22:00vs23:00h,whereassamples cortisolor we foundnosignificantdifferenceinsalivary sampled attheirnormalbedtime( well belowtheupperlimitofnormalinhealthysubjects cortisolandcortisonelevels A recentstudyfoundsalivary it couldpotentiallyinducestressandraisecortisollevels. 00:00 hmightberatherlateforsomeindividuals;thus, diagnosing CS,andthus,itmeritsfurtherstudy( and after DST could potentially increase the accuracy of glucocorticoids, measuredbothatthediurnalnadir exclude thatasimplesequentialevaluationofsalivary cortisoneat23:00halone.However,salivary wecannot traditional LNSCalone,butnothigheraccuracythanthe had significantly higherdiagnosticaccuracy thanthe this studywefoundthatbothofthesecombinedtests test mightbesuperiortooneindividualsample.In both theeveningnadirandsuppressibilityinone levels measuredat23:00andaftertheDSTthat evaluates Alternatively, cortisol(orcortisone) thesumofsalivary Clinical Study ). Within thecurrentreferencegroup,wefound Previous studies have shown that cortisol levels It hasbeensuggestedthat,inadditiontothewell- The traditionalsamplingtimeforLNSCat23:00– 12 , 37 17 , , 38 38 , , 39 39 , , 40 N Bäcklundandothers 36 43 , ). Inthecurrentstudy, 41 , 44 , 42 , 45 ). We collected , 46 , 27 47 ). , 48 ,

reject individualswithnon-CShypercortisolaemic states, cortisone. Furthermore,wedidnotactivelyrecruitor cortisol and the impact of these factors upon salivary and largerstudiesareneededtospecificallyaddress of thereference population areconsidered exploratory cortisolandcortisoneinsubgroups regarding salivary few,common diseasesarevery analyses thesecondary subjects withdifferentBMI,tobaccohabitsand the subgroups investigated. However, as the number of therefore donotrecommendseparatereferencerangesfor these subgroups.Basedonthecurrentstudyfindings,we value for the entire reference population, including all of sensitivity andspecificity, basedonthe97.5thpercentile could bedifferentiatedfromreferencesubjectswithhigh Nonetheless, itshouldbenotedthatpatientswithCS may beexpected,dependingonthesamplingtimepoint. cortisone levelsinthesesubgroups,slightlyhighervalues Accordingly, cortisoland wheninterpretingsalivary higher estrogenload,i.e.OC usersandpregnantwomen. cortisolandcortisoneinwomenwith for highersalivary OC-using premenopausalwomen. Therewasalsoatrend significantly higherplasma cortisolafterDSTthannon- this study, thefive premenopausalwomentakingOChad methodological variations( plasma cortisol levels with estrogen loads – albeitwith studies haveshownelevationsinbothLNSCandfree be unaffectedbyOCandpregnancy. However, some measurements should the freefractionofcortisol,salivary using OC( HPA-axis abnormalitiesinwomenthatarepregnantor could causedifficultiesininterpretingdiagnostictests for with concomitantincreasesintotalplasmacortisol.This glycyrrhizic acid( or inhibition,becausetobaccosometimescontains ratio couldbeduetoeither11 caused abstinence-relatedstress.Moreover, theincreased one hourpriortosampling,whichcouldhavepotentially sampling instructions,whichdidnotallowsmokingfor increased morning levels in smokers might be due to the cortisol:cortisone ratio were elevated in smokers. These cortisolandcortisonelevelsthe 08:00 hsalivary collected at23:00horafterDST, butspontaneous found nosignificanteffectsoftobaccouseinsamples are neededfortheseimportantgroupsofpatients.We cortisol and cortisone and cut-off values for salivary needed toestablishwhetherseparatereferenceintervals (i.e. pseudo-Cushingsyndromes).Furtherstudiesare including depression,alcoholabuse,andsevereobesity higher LNSCinoldervsyoungerhealthysubjects( diagnosis Salivary cortisoneinCushing Oestrogen isknowntoincreasecirculating CBGlevels, 3 , 16 , 18 6 , , 9 50 ). ). Becausesalivasamplesrepresent Downloaded fromBioscientifica.com at09/25/202110:23:41PM 4 , β 16 https://eje.bioscientifica.com -HSD2 enzymesaturation , 18 182 , 50 :6 , 51 , 52 , 53 579 ). In 49 via freeaccess ). European Journal of Endocrinology https://eje.bioscientifica.com Hospital, for the skilful inclusion of reference individuals and for handling for and individuals reference of inclusion skilful the for Hospital, Many thanks to Katarina Iselid at Clinical Research Centre, Umeå University Acknowledgements on pituitarydisease. research for University Umeå to donation a by supported was study This Funding be could that interest of conflict perceived asprejudicingtheimpartialityofthisstudy. no is there that declare authors The Declaration ofinterest EJE-19-0872 at paper the of version online the to linked is This Supplementary materials preanalytical errors. conjunction withfavourablerobustnesstoputative cortisol,in higher diagnosticaccuracythansalivary cortisoneshowingaslightly,salivary butsignificantly and afterDSThadahighdiagnosticaccuracyforCS,with cortisolandcortisoneat23:00 h Salivary from 22:00 h. indicatingthatLNSCmaybecollected 22:00 and23:00 h differences were found between samples collected at measured at23:00handafterDSTwithLC-MS/MS.No cortisone, cortisolandsalivary values forsalivary including patientswithadrenalincidentalomas. markers should also be tested in other relevant groups, cortisolandcortisonefordetectingCS.These salivary sensitivityandspecificityof to validatetheobserved assessed beforeexclusion( andalloutlierswerecarefully in theclinicallaboratory defining, establishing,andverifyingreferenceintervals was madeinaccordancewiththeCLSIguidelinesfor samples notconsideredrepresentative.Thecurrentstudy relevant referencesubjectsandexclusionof includesacarefulchoiceof relevant referenceintervals 70 yearsofageandhavediabetes).Establishingclinically reference subjectswithhypertensionmightalsobeover population, andtheoverlapbetweensubgroups(e.g. included thesmallsubgroupswithinreference spanning frommildtosevereCS.Thestudylimitations in areasonablylarge,representativegroupofpatients, adult population.We alsoevaluatedthedifferenttests large referencecohortthatwellrepresentthegeneral studies. However, thiswasnotsignificantandwarrantsfurther Clinical Study In conclusion,wehaveestablishedrobustreference A strength of ourstudywastheprospectively collected . 19 ). Futurestudiesareneeded N Bäcklundandothers https://doi.org/10.1530/ References analysis ofplasmadexamethasone. Steinar and Hustad, at the Core Facility for Metabolomics, University of Bergen, for the Bifulco Ersilia thank they Additionally, advice. Eriksson statistical Marie for and Lundquist Anders thank also authors The samples.

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Accepted 26March2020 Revised versionreceived4March2020 Received 29October2019

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