Copyright© ABE&M todos os direitos reservados. Accepted on Aug/2/2010Accepted Received onJan/5/2010 [email protected] 04039-032 −SãoPaulo, SP, Brasil 13º andar Rua Pedro de Toledo, 781, Universidade Federal deSãoPaulo deMedicina, Departamento de Endocrinologia, Laboratório deEsteroides,Divisão Claudio E.Kater Correspondence to: 3 2 1 B152TT, UnitedKingdom Biomedical Research, Birmingham, of Birmingham,Institute of Medical Sciences,University Oakland, CA,USA, Divisionof Oakland Research Institute, Dallas, Texas, UnitedStates Southwestern MedicalCenter, Sciences, Universityof Texas São Paulo, SP, Brazil Federal deSãoPaulo (Unifesp), of Medicine,Universidade Department and Metabolism, Unit, DivisionofEndocrinology 826 article original Children’s of Hospital DepartmentofClinical Adrenal andHypertension

Congenital adrenal hyperplasia; CYP17; 17-hydroxylase deficiency; 17,20-lyase deficiency; urinary steroidmetabolome; Congenital adrenalhyperplasia;CYP17;17-hydroxylasedeficiency;17,20-lyaseurinary Keywords Endocrinol Metab. 2010;54(9):826-32 urinários; corticosterona Hiperplasia adrenalcongênita;CYP17;deficiência de17-hidroxilase;deficiência17,20-liase;metabolomaesteroides Descritores Metab. 2010;54(9):826-32 ne, , 5-pregnenediol,17OH- andpregnanetriol. 18OH-DOC, and17OHP; andtetrahydro (TH)-B, THA, THDOC, THF+5a U samplesdrawntomeasure: (F),corticosterone (B),deoxycorticosterone (DOC),18OH-B, W406R orR362C;11 controlswereCYP17 wildtypes(WT). WT andcCYP17D patients hadSand ; and(3)comparedatafromthetwomostprevalentmutationsinBrazil. in vivo ciency (cCYP17D); (2)analyze17 α therelative 17OH-pregnenolona epregnanetriol. tetraidro(TH)-B,THA,TH-DOC,THF+5α e dosagem de:cortisol (F),corticosterona (B),deoxicorticosterona (DOC),18-OH-B, 18OH-DOC e17OHP; controles eramCYP17 wildtypes (WT). Amostras deSeUforamcolhidasdos WT epacientespara dos paraaDcCYP17, deumacoorte anterior, eramhomozigotos para W406R ouR362C(8cada);11 thods: patients withCYP17deficiency by urinary metabolomes of and 17,20-lyase activitiesrevealed Distinctive pr Marcos S. Neres depacientesurinários comdeficiênciadeCYP17 17,20-liase reveladas pormeiodometaboloma deesteroides Perfil característico dasatividades17-hidroxilase e Objetivos: RESUMO Objectives: ABSTRACT comparar asduasmutaçõesmaisprevalentesnoBrasil. da17(DcCYP17);α (2)analisarasatividades was alsoimpairedincCYP17D. W406 andR362CmutationsdisclosesimilarSand Upatterns. cretion of17-deoxysteroids, reductionofFand andmarked AA. Inadditionto17OH, 17,20-L activity na DcCYP17. As mutações W406 eR362Capresentamachados semelhantes emSeU. traste comareduzidaprodução/excreção deFe AA. da17OH e17,20-LAs atividades estãodiminuídas DcCYP17, aproduçãoeexcreçãodos17-deoxiesteroides estãoaumentadasemparalelo,con- W406R andR362Cmutations. that both17OH and17,20L wereimpairedincCYP17D. activities There werenodifferences between and adrenalandrogens(AA)werereduced;UsteroidsparallelSfindings.Metaboliteratiosrevealed WT, elevationsofB,DOC,18OH-B cCYP17D and18OH-DOC, patientshadmarked whereas17OHP, F DcCYP17. Nãohouvediferençasentrepacientescomasmutações W406R eR362C. de 17OHmetabólitos mostraram e17,20Lque as atividades estavam reduzidas em pacientes com adrenais (AA) estavam reduzidos. Os esteroides na U acompanham os achados no S. As relações de revelaram elevaçõesacentuadasdeB,DOC,18OHB e18OHDOC, enquanto17OHP, Feandrógenos 20genotypedcCYP17D patientsfromapreviouslyreported cohort werehomozygousfor (1) Caracterizar os esteroides séricos (S) e urinários (U) na deficiência completa da CYP17 (1) Caracterizarosesteroidesséricos(S)eurinários(U)nadeficiênciacompletadaCYP17 (1) Characterize serum(S)andurinary(U)steroidmetabolitesincompleteCYP17 defi- 1 , Richard J. Auchus Conclusions: ofile of the 17-hydroxylase Resultados: -THF, THE, androsterona, etiocolanolona,5-pregnenediol, cCYP17D patients show paralleloverproduction/overex -hidroxilase (17OH) e17,20-liase (17, ; e(3) 20 L)invivo 2 , H. Cedric L. Shackleton -hydroxylase (17OH) and17,20-lyase (17,20L) activities Comparados aos WT, ospacientescomDcCYP17 Sujeitos emétodos: Arq Bras Metab. Endocrinol 2010;54/9 -THF, androstero- TH-, 3 , ClaudioE. Kater 20pacientesgenotipa- Results: Subjects andme- Conclusões: Arq BrasEndocrinol Compared to Comparedto Arq Bras Na Na 1 - the Mennonitedescendants asamong orgeographicalareas, specific ethnicgroups perplasia (CAH).However, itisnotsouncommonin Arq Bras Metab. Endocrinol 2010;54/9 1 INTRODUCTION metabolite ratiosinpatients withcomplete17OHD bothenzymaticactivities,(2)compare ces concerning - to:(1)definepossibledifferen tabolite ratiosinorder tients and examined specific precursor-to-product me- of17OHDpa- metabolomeinalarge group steroid patients islimited. metabolite(metabolome)inthese steroid full urinary sively studiedin17OHD,systematicevaluationofthe hasbeenexten- profile steroid 25). Althoughtheserum andhypokalemia(2,21- hypertension causing severe activity −, withmineralocorticoid intermediates steroid (B)−non-17-hydroxylated (DOC) andcorticosterone ofdeoxycorticosterone stimulates overproduction that ACTH secretion 17OHD −,leadstoincreased −typicalofcomplete ofcortisol production impaired inthemale(46,XY)(17-20). Inaddition, phroditism - inthefemale(46,XX)andpseudoherma amenorrhea insexualinfantilism andprimary these patientsresults deficiency. in sexsteroids toproduce Thus,failure combined orcomplete17α-hydroxylase/17,20-lyase in domain resulting occurs inthesteroid-binding mutationsinCYP17(16) Most ofthe75reported b5(7,13-15). andtheco-factorcytochrome -reductase P450oxido- protein tion withtheelectron-transfer sitedomainforinterac- partner-binding tional redox sitedomain andafunc- functional steroid-binding a testes. Formaximalactivities,theenzymerequires ovariesand zonareticulata, intheadrenal precursor toyieldDHEA, anandrogen cleavage of17OHPreg ly, theC17-20side-chain thelatterperforms whereas - (17OHP),respective and 17α-hydroxyprogesterone (17OHPreg) to17α-hydroxypregnenolone terone - andproges pregnenolone outbyconverting carried activityis tivities, amongothers(4-12).Theformer and17,20-lyaseac- displaying both17α-hydroxylase andthegonads cortex intheadrenal me expressed P450c17,anenzy- gene whichencodescytochrome all cases(2,3). ofCAH,accountingfor5%-7% form most frequent thesecond 17OHDisconsidered and inBrazil,where In this paper we determined serum levelsandthe serum In thispaperwedetermined The diseaseiscausedbymutationsintheCYP17A1 considered a rare cause of congenital adrenal hy- causeofcongenitaladrenal arare considered deficiency(17OHD)isgenerally 7-Hydroxylase

of DutchFrieslanders(1) and theR362C. CYP17 mutationsdocumentedinBrazil,theW406R betweenthe twomostprevalent the metabolicpattern lated 17,20-lyasedeficiency(26),andalso(3)compare patients withiso- reported previously with thosefrom pants. - allpartici writtenconsentobtainedfrom informed ofourinstitutionand Committee onHumanResearch before. them hadneverbeentreated Fiveof profile. steroid theirpre-treatment to reestablish their medicationsdiscontinuedfor72h,inanattempt therapyhad replacement on ongoingglucocorticoid Allpatients . ning bloodsamplingforserum metabolitesandamor steroid samples) tomeasure collections(mostlyspoturine and includedurinary mutations inanyofthealleles(“wildtypes”). with 17OHD, but whose genotyping did not identify selectedamongthefamilies ofpatients yr). Theywere (5 male; 6 female, 15 to 57 years of age; median of 26 Y329X andW406R+M1T)(2). (R362C, W406R,R362C+W406R,Y329D+splice, genotyped and specific CYP17 mutations identified Allhadbeen withlowtoabsentsexsteroids. gesterone, - activityandelevatedLH, FSH, andpro plasma renin and , cortisol, values,asreduced laboratory lemia. Complete17OHDwasestablishedbytypical andhypoka- ofhypertension ciated withthepresence periods at thetime of puberty,menstrual mostly asso- sexcharacteristicsandof by theabsenceofsecondary of 22yr);inmost,clinicaldiagnosishadbeensuspected dy, 14to42years(median patientsranged in agefrom (2).Atthetimeofstu- reported patients previously of alarger cohort selectedfrom notypic female)were ofCAH(twelve46,XY,form andeight46,XX,allphe- withthecomplete17OHD Twenty patients affected SUBJECTS ANDMETHODS following serum steroids were measured intheClinical measured were steroids following serum untiltakentothelaboratory.under refrigeration The (a fewcollected24-hoururine specimens)andkeepit sample tocollectaurinary instructed drawn andwere blood sample hadamorning All patientsandcontrols Analytical methods The study protocol was previously approved bythe approved waspreviously The studyprotocol inallsubjects wasperformed The studyprotocol alsostudiedascontrols subjectswere Eleven normal Steroid metabolites inCYP17deficiency 827 -

Copyright© ABE&M todos os direitos reservados. Copyright© ABE&M todos os direitos reservados. Steroid metabolites inCYP17deficiency 828 etiocholanolone. 17-hydroxypregnenolone; PT: ;THF:;5a-THF:allo-tetrahydrocortisol;THE:; AN:;ET: tetrahydrocorticosterone; THA:tetrahydro-11-dehydrocorticosterone; 5a-THA:allo-tetrahydro-11-dehydrocorticosterone; 5-PT: 5-pregnenetriol;17HP: parenthesis). 5PD:5-pregnenediol; PD: ;THDOC:tetrahydro-11-deoxycorticosterone;THB: tetrahydrocorticosterone; 5a-THB:allo- steroidmetabolites(inboldand where 17-hydroxylaseand17,20-lyase operate,theadditionalhydroxylationsteps(initalic),andrespective urinary Figure 1.Schematicillustrationoftheadrenocortical steroidbiosynthesis,identifyingsteroidsproducedinthedelta-5and in thedelta-4-pathways,sites - andros metabolitesofsexsteroids: well astheurinary as (THE)(metabolitesofcortisol), trahydrocortisone (THF)and 5a trahydrocortisol te- (PT) (metabolitesof17-hydroxyprogesterone); (17HP)andpregnanetriol 17-hydroxypregnenolone pound A(THA)and5a orcom- plus tetrahydro-11-dehydrocorticosterone (THB)and5a tetrahydrocorticosterone (THDOC)(metabolite ofDOC); tetrahydro-DOC respectively); andprogesterone, sors, pregnenolone precur (metabolites ofthenon-17-hydroxylated red: specific enzymaticactivities(27). calculatedtoexpress ratioswere and specificsteroid asµg/Lofurine) quantified (concentrationsexpressed identifiedand were analysis. Thecompoundsofinterest spectrometry (CHLS) forgaschromatography-mass Institute HospitalofOaklandResearch the Children’s subsequentlymailedto were te extraction.Cartridges metaboli- forsteroid aC-18cartridge passed through 17OH-. and aldosterone, (18OHB), 18OHDOC,cortisol, (DOC),18-hydroxyB (B),deoxycorticosterone rone - radioimmunoassay afterHPLCseparation:corticoste Center at SanFrancisco General Hospital by Research The following urinary metabolites were measu- metaboliteswere The followingurinary specimen,a20mLaliquot was eachurinary From D 5-pregnenediol (5PD) and pregnanediol (PD) (5PD)andpregnanediol 5-pregnenediol D4-Pathway: PROGESTERONE→17a-Hydroxylase-OH-PROGESTERONE17,20-Lyase → D5-Pathway: PREGNENOLONE→17a-Hydroxylase-OH-PREGNENOLONE17,20-Lyase →DHEA

DEHYDROCORTICOSTERONE (THA,5a-THA=As) (THB, 5a-THB=Bs) ↓ 11b-Hydroxylase CORTICOSTERONE ↓ 21-Hydroxylase

↓ 11b-HSD2 ↓ 3b-HSD2

(allo)-THA (metabolites of B); (allo)-THA (metabolitesofB); (THDOC) (5PD) (PD) DOC (allo)-THF plus te- (allo)-THB (5-PT, notmeasured) (THF, 5a-THF=Fs) 11-DEOXYCORTISOL - ↓ 11b-Hydroxylase ↓ 21-Hydroxylase ↓ 11b-HSD2 (17HP, PT) CORTISONE ↓ 3b-HSD2 CORTISOL (THE=E) ted 17,20-lyasedeficiency”(26). publicationthatstudiedpatients with“isola- previous a ourpatientswiththosefrom datafrom to compare (AN+ET) indicatespecifically17,20-lyaseactivity. (3) (17HP+PT)/(AN+ET),PT/(AN+ET),and(Fs+E)/ solely;and, of17-hydroxylation stand fortheefficiency (5PD+PD)/(17HP+PT), plus17,20-lyase);(2) CYP17 activity(17-hydroxylase ofglobal usedasexpressions (Bs+As)/(AN+ET) are (1)(5PD+PD)/(AN+ET)and and “E”,respectively: as“Bs”,“As”,“Fs”, and THE,willbenamedherein tes: (THB+5a Forsimplicity,1). ofmetaboli- thefollowinggroups gure the specific precursor-to-product metaboliteratios(Fi- of androstenedione). (AN)plusetiocholanolone(ET)(metabolites terone were alsotypicallylow(datanotshown). were androgens levels. Adrenal aldosterone to reduced ble with normal versely, undetecta- virtually and 17OHP were cortisol toWT(Table ascompared and progesterone, 1).Con- B,DOC,18OHB, 18OHDOC to >100-fold)ofserum Patients with17OHDshowedmarkedelevations(20- RESULTS The underlined ratios shown above were employed The underlinedratiosshownabovewere Enzymatic activities were inferred from calculation of calculationof from inferred Enzymatic activitieswere THB), (THA+5a PD/PT Arq Bras Metab. Endocrinol 2010;54/9 (AN, ET) THA), (THF+5a , and (Bs+As)/(Fs+E) , and(Bs+As)/(Fs+E) THF), THF), sed (Figure 2). In contrast, cortisol metabolites,THF, 2).Incontrast,cortisol sed (Figure - alsosignificantlyincrea (5PDandPD)were gesterone - andpro substrates, pregnenolone the 17-hydroxylase 2).Accordingly,with THDOC(Figure metabolites of inparallel significantlyincreased, cially 5aTHBwere tion ofBmetabolites:THA,5aTHB,andspe- - findings.Excre withtheir serum concordant mes were Arq Bras Metab. Endocrinol 2010;54/9 of corticosterone:THB, 5α-THB,THA,-THA. metabolites of pregnenolone (Pregnen), progesterone (Prog) and deoxycorticosterone (DOC): 5-PD, PD, andTHDOC. Right-hand panel– steroid metabolites Figure 2. steroidlevelsfrom20patientswith17OHD(darkcircles)and11normal controlindividuals(lightcircles).Left-handpanel–steroid Urinary ** Normalrangesformaleandfemalecontrols(atthefollicularphase)obtainedfromref.28. * Referencevaluesobtainedfromref.5. disease (allgiveninng/dL,exceptforcortisol,μg/dL) inpatientswith17OHDand“wildtypes”(WT)forthe interquartile interval) Table 1.Basalserumsteroidlevels(mean±SEandmedianplus B Steroid DOC 18OHB 18OHDOC Cortisol Progesterone 17OHP Aldosterone In 17OHD patients, the urinary steroid metabolo- steroid In 17OHDpatients,theurinary 14,800 (4,749-32,900) 865 (590-1,330) 16,299 ±1,488 325 (120-921) 278 (169-635) 276 (126-704) 2.6 (0.2-11.0) 0.2 (0.2-0.5) 2.5 (1.0-3.0) (n =20) 323 ±42 312 ±40 306 ±36 0.3 ±0.0 905 ±94 2.3 ±0.3 3.8 ±0.8 17OHD

≤10 Pregnen./ Prog./ DOC metabolites (μg/L) 10 10 10 10 2 3 4 5 (30-150 /30-70)** 22.1 (10.0-39.7) 129 (55-1,557) 10.4 (4.3-25.7) 5-PD 4.4 (0.4-10.9) 3.1 (2.0-49.4) 5.1 (2.0-10.6) 23.5 ±2.4* 84 (6-215) 358 ±150 10.9 ±5.2 11.6 ±1.8 (n =11) 4.3 ±1.0 4.6 ±0.8 90 ±23 WT PD THDOC < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 NS p (around 400-foldhigherthanWT)(26). (around intermediate “isolated 17,20-lyasedeficiency”were forthelatterratioinpatientswith The valuesobserved activities)(Table17α-hydroxylase/17,20-lyase 2). totalCYP17deficiency(combined ofvirtually result toWT,fold) in17OHDpatientsascompared asa significantlyelevated(4,500-to8,500- ratios were The (5PD+PD)/(AN+ET)and(Bs+As)/(AN+ET) Ratios 3). plus PTandANET(Figure 5aTHF, aswell17HP decreased, andTHE,were lar andnotstatisticallydifferent. simi- ratios were metabolite valuesandtheirrespective allindividual compared, and R362Cmutationswere ratio isanalyzed(8.37[3.44-13.38]vs.6.63[3.88-14.94]). itwassimilarwhenthe(Fs+E)/(AN+ET) 4.30]), whereas 17,20-lyase deficiency”(0.84[0.64-1.23]vs.2.25[1.72- in ourpatientswith17OHDthanthose“isolated 17,20-lyaseactivitywassignificantly lessimpaired dered, WT, WhenthePT/(AN+ET)ratioisconsi- respectively). 17,20-lyase deficiency”(1.7-foldand37-foldhigherthan inpatientswith“isolated rately (Bs+As)/Fs+E)affected activitywasslightly(PD/PT) tomode- 17-hydroxylase (Fs+E)/(AN+ET) (4-to10.5-fold)(Table 2).However, to(17HP+PT)/(AN+ET) and (> 400-fold),ascompared (5PD+PD)/(17HP+PT) and (Bs+As)/Fs+E) ratios ty, in the increase by the disproportionate as revealed over17,20-lyaseactivi- affected was predominantly Corticosterone metabolites (μg/L) ≤10 When examinedinseparate,17α When homozygote17OHDpatientsfortheW406R 10 10 10 10 2 3 4 5 THB 5a-THBTHA-THA Steroid metabolites inCYP17deficiency -hydroxylation -hydroxylation 829

Copyright© ABE&M todos os direitos reservados. Copyright© ABE&M todos os direitos reservados. 830 tobe 17OHD, in whom enzymatic activity was proven 20patientswith metabolomesfrom steroid the urinary levels and steroid In thisstudyweexaminedthe serum DISCUSSION * Ratiosderivedfromdatareportedinref.26. + 5a(allo)-THF;E:THE(tetrahydrocortisone). 5a(allo)-THA; 17HP:17-hydroxypregnenolone;PT: pregnanetriol);Fs:THF(tetrahydrocortisol) (tetrahydrocorticosterone) +5a(allo)-THB;As:THA(tetrahydro-11-dehydrocorticosterone) 5PD: 5-pregnenediol;PD:pregnanediol;AN:androsterone;ET: etiocholanolone;Bs:THB “wild types”(WT)forthedisease patients with17OHD,“isolated17,20-lyasedeficiency”(17,20-LD*)and in steroidmetabolites (median plusinterquartile interval) ratios ofurinary Table 2.Relative enzymatic activities of CYP17 as determined by diagnostic of 17-OH-progesterone(17HPandPT)androstenedione(ANET).Righthandpanel–steroidmetabolitescortisol:THF, 5α Figure 3. steroidlevelsfrom20patientswith17OHD(darkcircles)and11normalcontrolindividuals(lightcircles).Lefthand panel-steroidmetabolites Urinary Steroid metabolites inCYP17deficiency metabolite ratios steroid Urinary (5PD+PD)/(AN+ET) CYP17 (17-hydroxylase+17,20-Lyase) (Bs+As)/(AN+ET) (5PD+PD)/(17HP+PT) 17-hydroxylase activity PD/PT (Bs+As)/(Fs+E) (17HP+PT)/(AN+ET) 17,20-lyase activity PT/(AN+ET) (Fs+E)/(AN+ET)

≤ 1 17OH-Progesterone / A’dione metabolites (μg/L) 10 10 10 10 (348.3-1,136.8) 4 1 3 (130.3-315.8) (139.0-296.8) (107.8-226.4) (63.7-114.4) (3.44-13.38) (0.77-1.48) (0.64-1.23) (n =20) 17-HP PT 17OHD 243.6 714.5 219.3 149.2 94.7 1.04 0.84 8.37 (3.88-14.94) (29.6-67.4) (0.57-1.15) (5.04-9.66) (1.72-4.30) 17,20-LD* (n =6) 58.4 0.74 8.07 2.25 6.63 – – – AN (0.10-0.22) (0.11-0.28) (0.42-0.74) (0.35-0.64) (0.14-0.24) (0.21-0.29) (0.17-0.23) (0.62-1.26) (n =11) ET 0.14 0.15 0.53 0.43 0.22 0.27 0.20 0.80 WT

Cortisol metabolites (μg/L) ≤ 1 10 10 10 10 1 2 3 4 vated, mostly after ACTH stimulation, but suspicion is vated, mostlyafterACTHstimulation, butsuspicionis is alsoele- In isolated 17,20-lyase deficiency progesterone evidenceforthediagnosisof17OHD. a simpleyetstrong hypogonadismis ofhypergonadotrophic in thepresence and cols.(28)whosuggested thatelevatedprogesterone byMartin datareported found inourpatientscorroborate increase zymatic deficiencies.Themarkedprogesterone gender, en- elevated in other adrenal and also frequently of inpatientswith17OHDregardless variably increased sor andthemajordelta-4substrateforCYP17.Itisin- ble 18OHB contribution from thiszone). ble 18OHBcontributionfrom in17OHD(togetherwithanegligi- ristically reduced ischaracte- aldosterone zonaglomerulosa suppression, renin withhypokalemiaandchronic coid hypertension - inthepathogenesisof mineralocorti play amajorrole (22,23).Sinceelevated DOCandB ACTH production stimulatedbyincreased corticosterone, dant precursor itsabun- tion of18OHBinthezonafasciculatafrom - auniquefindingexplained bytheproduc aldosterone, ofreduced inthepresence sor inthezonaglomerulosa) precur tween theraisedlevelsof18OHB(aldosterone thway”) ofthezonafasciculata. pa- the“ (from 17-deoxysteroids -derivatives –18OHBand18OHDOC–,theso-called - ofB,DOCandtheir 18-hydroxy marked increases published (8,20,24,25,28) and disclosed se previously (2). elsewhere tations W406RandR362C,asreported completely abolishedbythespecificCYP17A1genemu- THF 5a-THFTHE Progesterone is an earlier steroid biosyntheticprecur isanearlier steroid Progesterone in this study is the dissociation be- Also confirmed wascomparabletotho- profile steroid The serum Arq Bras Metab. Endocrinol 2010;54/9 -THF andTHE. - - Arq Bras Metab. Endocrinol 2010;54/9 similar enzymaticfunctions.Usingthissetof express metabolite ratios which, nevertheless, ghtly different have isolated17,20-lyasedeficiency, weexaminedsli - cols. (26)whostudiedpatients initiallyassumedto ofdeficient 17-hydroxylation. asaresult reduced (17,20-lyase)wouldbe substrate forthissecondreaction ratios forthelatterisnotsurprisingsinceamountof xylase obliteration. The lower yet significant increased - 17-hydro complete 17OHD, denoting a predominant than (400- to900-foldand3-10-fold,respectively) of magnitude at less least one and two orders they were metaboliteratios);however, ofproper ciencies (increases enzymatic defi- patients, also demonstrates expressive of 17a complete lossofCYP17function.Aseparateevaluation of 4,000-to8,000-foldhigherthanWT–,typicala highandunequivocalvalues–intherange showed very mate globalenzymaticactivityinpatientswith17OHD diagnosis ofseveralbiosyntheticdefects(26,27). usefulforthe tios. Thisinvivoassessmenthasproven analyzing specificprecursor-to-product metabolitera- estimated by and 17,20-lyase) were (17-hydroxylase alsosubstantiallyreduced. and PT),are (17HP te metabolites,especially17OH-progesterone - intermedia duction. Inaddition,the17-hydroxylated - absentpro theirvirtually complete 17OHD,reflecting in patients with reduced appreciably metabolites are inthesepatients. tive, ifnotincreased, 5 thepreferential Nevertheless, processing. ven excretory dispersion intoseveralothermetabolitesand/oranune- gher thanWT, thebroad possiblyreflecting respectively), levels(25-foldvs.65-foldhi- than thatseenwithserum andlessexpressive issomewhatdisproportional se steroid of the- THDOC. The magnitudeof metabolite excretion (THA, 5a (THB,5a the reduced in 17OHDpatients.Also,bothBmetaboliteisomers, substantiallyincreased (5PDandPD),are progesterone and suchaspregnenolone early biosyntheticsteroids, metabolitesof Urinary mixture. tabolism ofthesteroid andperipheralme- hepatic,renal plying anappropriate findings, withserum inagreement this studywere im , assuggestedbyAuchusandMiller(6). increased ratiois best raisedwhenthe17OHPtoandrostenedione a -reduction catabolic pathway remains functionallyac- catabolicpathwayremains -reduction For comparison with the report byTiosanoand For comparisonwiththereport The precursor-to-product metaboliteratiosthatesti- enzymaticactivities of CYP17 The relative andandrogen On theotherhand,bothcortisol metabolomesevaluated in steroid The urinary -hydroxylase and 17,20-lyase activities in the same and17,20-lyaseactivitiesin thesame -hydroxylase THA) forms, are distinctly elevated, as well as distinctlyelevated,aswell are THA) forms, THB) and the dehydrogenated THB) andthedehydrogenated - between groups. tic activity, didnotevidenceanysignificantdifferences with 17OHD,bothknowntoabolishCYP17enzyma- mutations inBrazilianpatients prevalent the twomore type sometimesassociatedtothesamemutations. and characterize the variability of the individual pheno- influence ofmultiplemodulatorsormodifyingfactors underthe are sumption thatmanyenzymaticprocesses expression enzymatic thanthose obtainedfrom precise seem more theseinvivostudies and cols.(26),dataderivedfrom byTiosano 17OHD patients.However, asobserved and17,20-lyase activitiesin of absent17-hydroxylase invitro previous studied inthispaperconfirm findings (30). in thegeneofP450oxidoreductase showed a homozygous mutation G539R reinvestigation diagnosedashaving17,20-lyase deficiency,previously systematic metabolic studies.Also, inatleastonefamily isolated17,20-lyasedeficiency (14,15)precludes “true” CYP17A1mutations,sincetherarityof ofdifferent result asa spectrum both activitiesmayhaveanimpairment gh shown whom theE305GmutationinCYP17A1genewas byTiosanoin reported “isolated 17,20-lyasedeficiency” deficiency”,inanalogy tothosewith lated 17-hydroxylase patients with“complete17OHD”mayinfacthave“iso- withtheFs+E/AN+ETratio. be observed could 17OHD patients.Similarbutlessevidentresults inTiosano’sthanour products 17-hydroxylated availabilityof agreater xylase, orinotherwords, - of17,20-lyase over17-hydro dominant impairment comparison withWT,- indicatingthepre respectively), Tiosano’s patientsthaninours(11-foldvs4-fold ratio wastheonlyonethatdiscloseshighervaluesin deficiency, madebytheauthors. already anobservation fold higherthanWT, 17-hydroxylase indicatingapartial values1.7-to37- sano andcols.(26),theseratiosreach byTio- However,former). inthesixpatientsreported to WT (~400-foldhigherinthe patients as compared for17OHD findingsobserved theprevious confirms patients was12-foldhigherthanintheirs. fold higherthaninWT, theBs+As/AN+ETratioinour in Tiosano’spatientsthanours:althoughnearly400- data, globalenzymaticactivitywasmuchlessaffected in vitro activity(29).Indeed, studiesindicatenormal nalysis from dataanalysisfrom In spiteoftheseobservations, metabolome and the metabolite ratios The urinary itistempting tospeculatethatsome In thisregard, 17,20-lyaseactivity,Concerning thePT/(AN+ET) activityalonealso Analysis ofthe17-hydroxylase to affect also 17-hydroxylation, althou- also17-hydroxylation, in vivotoaffect (26). This observation allowstheas - in vitro (26).Thisobservation Steroid metabolites inCYP17deficiency 831

Copyright© ABE&M todos os direitos reservados. Copyright© ABE&M todos os direitos reservados. of serum steroids and urinary metabolites. andurinary steroids of serum 17OH activity, pattern disclosed asimilarlyabnormal tions W406andR362C,bothassociatedwithabsent 17OHD. HomozygotepatientsfortheCYP17muta- incomplete tion, 17,20-lyaseactivityisalsoimpaired - Inadditionto17-hydroxyla androgens. and adrenal ofcortisol andexcretion intheproduction decrease thway ofzonafasciculata”),incontrastwithamarked pa- (“themineralocorticoid 17-deoxysteroids cortical - of the adreno and overexcretion rallel overproduction 832 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. REFERENCES was reported. relevant tothisarticle nopotentialconflictofinterest Disclosure: assembling alltheurinesamplesusedinthisstudy. Acknowledgements: We thankDr. MarivâniaCosta-Santosfor Steroid metabolites inCYP17deficiency

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