C-type natriuretic peptide plasma levels are reduced in obese adolescents

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Peptides

jo u r n al h om ep age :w w w . e l s e v i e r . c o m / l o c a t e / p e p t i d e s

C-type

natriuretic

peptide

plasma

levels

are

reduced

in

obese

adolescents

S.

Del

Ry

_,

_M.

_Cabiati

_,

_V.

_Bianchi

_,

_S.

_Storti

_,

_C.

_Caselli

_,

_T.

_Prescimone

_,

_A.

_Clerico

_,

G.

Saggese

_,

_D.

_Giannessi

_,

_G.

_Federico

a_{CNRInstituteofClinicalPhysiology,CNR,Italy}

b_{SezionediDiabetologiaPediatrica,U.O.PediatriaUniversitaria,AziendaOspedalieroUniversitariaPisana,Pisa,Italy} c_{FondazioneG.Monasterio,Pisa,Italy}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received22July2013 Receivedinrevisedform 24September2013 Accepted24September2013 Availableonline8October2013 Keywords:

C-typenatriureticpeptide Obesity

Adolescents Natriureticpeptides

a

b

s

t

r

a

c

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Thehighprevalenceofobesityinchildrenmayincreasethemagnitudeoflifetimeriskofcardiovascular disease(CD).Atpresent,explicitdataforrecommendingbiomarkersasroutinepre-clinicalmarkersof CDinchildrenarelacking.C-typenatriureticpeptide(CNP)isassumingincreasingimportanceinCD; inadultswithheartfailure,itsplasmalevelsarerelatedtoclinicalandfunctionaldiseaseseverity.We havepreviouslyreportedfivedifferentreferenceintervalsforbloodCNPasafunctionofageinhealthy children;however,dataonplasmaCNPlevelsinobesechildrenarestilllacking.Aimofthisstudywasto assessCNPlevelsinobeseadolescentsandverifywhethertheydifferfromhealthysubjects.PlasmaCNP wasmeasuredin29obeseadolescents(age:11.8±0.4years;BMI:29.8±0.82)byradioimmunoassayand comparedwiththereferencevaluesofhealthysubjects.BNPwasalsomeasured.BothplasmaCNPand BNPlevelsweresignificantlylowerintheobeseadolescentscomparedtotheappropriatereferencevalues (CNP:3.4±0.2vs13.6±2.3pg/ml,p<0.0001;BNP:18.8±2.6vs36.9±5.5pg/ml,p=0.003).Therewasno significantdifferencebetweenCNPvaluesinmalesandfemales.Asreportedinadults,weobservedlower plasmaCNPandBNPlevelsinobesechildren,suggestingadefectivenatriureticpeptidesysteminthese patients.Analteredregulationofproduction,clearanceandfunctionofnatriureticpeptides,already operatinginobeseadolescents,maypossiblycontributetothefuturedevelopmentofCD.Thus,the availabilityofdrugspromotingtheactionofnatriureticpeptidesmayrepresentanattractivetherapeutic optiontopreventCD.

©2013ElsevierInc.Allrightsreserved.

1. Introduction

Obesityistheresultofachroniccaloricimbalance,whenmore caloriesareassumedthanconsumedeachday.Mostobeseadults wereobeseasadolescentsandmostadolescentswereoverweight and/orobeseaschildren [40]; infact,theoriginsofobesityare beingtracedtoearlychildhooddevelopment.Childhoodobesityisa worldwidehealthproblemanditsprevalenceisincreasingsteadily and dramatically worldwide [44]. Obese children have a much greaterlikelihoodthantheirnormal-weightcounterpartsof acquir-ingdyslipidemia,hypertensionandimpairedglucosemetabolism, withanincreasedriskofdevelopingcardiovascularandmetabolic diseasesasadults[33].Childhoodobesityisalsoamajorriskfor earlyonsetofendothelialdysfunctionandatherosclerosis[30,48]. Atpresent,explicitdataforrecommendingbiomarkersasroutine

∗ Correspondingauthorat:CNRInstituteofClinicalPhysiology,ViaGiuseppe Moruzzi1,56124Pisa,Italy.Tel.:+390503152793;fax:+390503152166.

E-mailaddress:delry@ifc.cnr.it(S.DelRy).

pre-clinicalindicesofcardiovasculardisease(CD)inchildrenare lacking.C-typenatriureticpeptide(CNP),amemberofnatriuretic cardiac peptides, is assuming increasing importance in cardio-vascular disease. In adults a relationship betweenplasma CNP levelsandclinicalandfunctionaldiseaseseveritywasobserved, especiallyinheartfailure[10–15,20,22,35,46,49].Recently,five dif-ferentreferenceintervalsforbloodCNP,asafunctionofage,were reportedinhealthychildren[16].Inaddition,ithasbeenshown thatnatriureticpeptidesplay acentralrole intheregulationof bodyweightandenergymetabolism[32].Interestingly,ithasbeen shownthatANPandBNParesignificantlylowerinoverweightand obeseadultsubjectsthanintheleanones[8,18,21],probably con-tributingtoinsulinresistanceandhypertension.Dataonnatriuretic peptidesinchildrenofpediatricagearescarceandlimitedtoCNPin infancy[36–38],whiledataonplasmaCNPlevelsinobesechildren arestilllacking.

Aims of the present study wereto assess how plasma CNP levelsbehavein obeseadolescentscompared tonormalweight subjects,andwhethertheyshowthesamepatternobservedfor BNP and ANP in adult obese patients. To better describe the

0196-9781/$–seefrontmatter©2013ElsevierInc.Allrightsreserved.

Table1

Clinicalcharacteristicsofthestudiedsubjects.

Morphometricfeatures(cases,n=29)

Sex 17♂–12♀ Age(years) 11.77±0.4 Pubertalstage 1:n=7 2:n=12 3:n=5 4:n=4 5:n=1 Height(cm) 154.43±2.2 Weight(kg) 71.75±2.9 BMI 29.8±0.8 Obese/overweight Obese,n=25 Overweight,n=4 Fatmass,FM(%) 37.1±1.2

Fatmass,FM(percentile) >95,n=8

>98,n=21

Systolicpressure,SBP(mmHg) 109±2

Systolicpressure,SBP(percentile) 55.25±4.9

Diastolicpressure,DBP(mmHg) 65±1

Diastolicpressure,DBP(percentile) 52.7±3.7

neuroendocrineprofile,BNPwasalsomeasuredinthesame

sam-ples.

2. Methods

2.1. Subjectsandplasmacollection

Twenty-nine overweight/obese adolescents (17M, 12F, age

11.8±0.4 years), without cardiac dysfunction, referred as

out-patients to the Unit of Pediatric Endocrinology and Diabetes,

DepartmentofPediatrics,UniversityofPisa,Italy,wereenrolled

inthestudy.

Table1summarizestheclinicaldetailsofeachparticipantinthe study.BMI,calculatedusingtheformulaweight(kg)/height(m)2_, was29.8±0.82, correspondingto2.9±0.7 Z-scores[6]. Accord-ingtothedefinition oftheinternationalTask ForceonObesity inchildhoodandusingpopulationreferencedataspecificforage andsexforBMI[7],25/29wereobesewhiletheremaining4were overweight.Totalbodyfat,measuredusingtheTanitaBC-418 Seg-mental Body Composition Analyser (Tanita Corporation,Tokyo, Japan)showedthatourpatientshadFM%intherangeofobesity [27].

Bloodpressurewasmeasuredbythesameinvestigatorusinga standardvalidatedprotocol.Intwosubjectssystolicbloodpressure wasabovethe95thpercentileforheight,age,andsex-specific ref-erencevalues[45]indicatingaconditionofsystolichypertension.

Table2summarizesthelaboratorycharacteristicsofeach par-ticipantinthestudy.Fastingplasmaglucose(FPG)wasnormalin all.FifteensubjectsshowedHOMA-IR(HOmeostasisModel Assess-mentofInsulinResistance)values>2.0Z-scores,suggestingthat theyhadareducedinsulinsensitivity.Thisindexwascalculated accordingtheformula:fastingplasmainsulininuU/ml×FPGin mmol/L/22.5[26].Theresultswerecomparedwithreference val-uesspecificforItalianchildrenandadolescents,obtainedusingthe sameformula[9].

Regardingblood lipids,accordingtoreferencevaluesspecific forageandsex[43],fourpatientshadlevelsoftotalcholesterol abovethe95thpercentile;fourhadLDLcholesterolabovethe95th percentile,whiletryglycerideswereincreasedinsix.Bloodlevels ofHDLcholesterolwerelowerthan5thpercentileinfourpatients [43].

Bloodsampleswerecollectedinallthesubjectsby venipunc-ture, in the morning after an overnight fasting. In order to minimizedegradation,bloodsamplesforCNPwerecollectedin ice-chilleddisposablepolypropylenetubescontainingEDTA(1mg/ml)

Table2

Laboratoryfindingsofthestudiedsubjects.

Biochemical parameters

Case(n=29) Referencecut-off Glycemia(mg/dl) 72.5±1.9 <100mg/dl Insulin(uU/ml) 21.3±2.2 <15uUI/ml

HOMA-IR 4.0±0.4 Males:1.37±0.7 Females:1.65±1.1 HOMA-IR(Z-score) 2.9±0.5 – Cholesterol(mg/dl) 172±7 <180mg/dl(<75thpercentile) acceptable >210mg/dl(>95thpercentile) elevated HDL(mg/dl) 45±2 >37mg/dl(>5thpercentile) LDL(mg/dl) 107±5 <110mg/dl(<75thpercentile) acceptable >135mg/dl(>95thpercentile) elevated Triglycerides(mg/dl) 97±10 <110mg/dl(<75thpercentile) acceptable >170mg/dlinfemales(>95th percentile)elevated >150mg/dlinmales(>95th percentile)elevated

andaprotinin(500KIU/ml)topreventproteolysis.Sampleswere

rapidlyseparatedbycentrifugationfor15minat4◦C,andplasma

storedfrozenat−80◦_{Cin1-mlaliquotsinpolypropylenetubes}

untilassay,performedwithin1month.Bloodsamplesforblood

glu-coseandlipidsassayswerecollectedinlithium-heparincontaining

vials,whileinsulininEDTAcontainingvials.

Thestudywasconductedinaccordancewiththeguidelines

pro-posedintheHelsinkiDeclarationandapprovedbythelocalethics

committee.Informedconsentwasobtainedfromtheparentsof

eachsubject.

2.2. Biochemicalparametersassays

ACobasIntegra400analyser(Roche,Italy)andtheappropriate

commercialkitswereusedtomeasurebloodglucose(CobasIntegra

400GlucoseHK;enzymaticreferencemethodwithhexokinase),

totalcholesterol(CobasIntegra400Cholesterol;enzymatic,

colori-metricmethodwithcholesterolesterase,cholesteroloxidase,and

4-aminoantipyrine),HDLandLDLcholesterolfractions(Cobas

Inte-gra400HDL-CholesterolandLDL-Cholesterolplus2ndgeneration;

homogeneous enzymatic colorimetric assays) and tryglicerides

(CobasIntegra400Tryglicerides;enzymatic,colorimetricmethod

withglycerolphosphateoxidaseand4-aminophenazone).

Circulating insulin levels were measured by a commercial

immunoassaykit(Access® _{UltrasensitiveInsulin,Beckman}

Coul-terInc.,Fullerton,CA,USA),withasensitivityof0.03_␮IU/mLanda

precisionof<10%CV.

2.3. CNP,BNPassay

CNPwasdirectlymeasuredinplasmaby aspecific

radioim-munoassay (RIA) (Phoenix Pharmaceuticals, Belmont, CA,USA).

Eachsamplewasdeterminedinduplicateandtheassaywascarried

outonice.Acontrolsample,preparedbyusingknownamountsof

CNPaddedtotheassaybufferandstoredinaliquotsto−80◦_C,was

assayedineachrunforqualitycontrol.

As reported in a previous study of ours [16], the working

range was derived by the mean dose–response curve for CNP assayandthemeanimprecisionprofile,calculatedbyapreviously describedcomputerprogram[34];atthelowrange(CNP concen-tration:5–80pg/tube)theCV% resultedlowerthan20%.CNPin vitro-stabilitywasevaluatedmeasuringaplasmapoolindifferent experimentsperformedduring12monthsandthebetween-assay

Table3

ReferenceintervalsforCNPandBNPplasmalevelsinchildhood(modifiedbyRef.

[16]).

Agereferenceintervals CNP(pg/ml) BNP(pg/ml)

0–3days 11.6±2.1 396.2±100.3

4–30days 16.4±3.7 99.6±36.3

1–12months 15.4±2.7 29.2±3.92

1–12years 13.6±2.3 37.2±5.7

variabilityresulted<30%(n=7).Within-assayvariabilitywas

eval-uated using pools with different CNP concentration and both

resulted<20%:7.05±0.2pg/tube(n=9duplicateassays,CV=11%)

and 12.4_±1.1pg/tube (n=5 duplicate assays, CV=18%)pg/tube.

Analyticalsensitivityresulted0.77±0.05pg/tube.

PlasmaBNPwasmeasuredusingthefullyautomatedAccess

platform(TriageBNPreagents,AccessImmunoassaySystems,REF

98200; Beckman Coulter Inc., Fullerton, CA, USA). The

analyti-calcharacteristicsand performanceoftheAccessimmunoassay

methodusedinthisstudyformeasurementofBNPwere

previ-ouslyevaluated[5]aswellasthereferencerangesinchildhood

[39].

PlasmaCNPandBNPresultswerecomparedwithappropriate referencevaluesspecificforageandsex[16].InTable3the refer-enceintervalsforCNPandBNPplasmalevelsinchildhood,obtained inapreviousstudyofours[16],werereported.

2.4. Statisticalanalysis

AllsamplevaluesandotherdataforqualitycontroloftheRIA systemwere calculated using a previously described computer program[34];theinterpolationofthedose–responsecurveswas computedusingafour-parameterlogisticfunction[34].Because plasmaCNPvaluesarenotnormallydistributed,natural logarith-mictransformationofdatawasusedforstatisticalanalysiswhen needed.DifferencesofCNPlevelsbetweentwoindependentgroups wereassessedbyunpairedt-test.Relationsbetweenvariableswere assessedbylinearregression.Resultsareexpressedasmean±SEM andap-value<0.05wasconsideredsignificant.

3. Results

Plasma CNP resulted significantly lower (p<0.0001) in the obesesubjectsincomparisonwiththeappropriatereference val-ues(Fig. 1a)[16].BNP plasmalevels werealsocompared with

theappropriatereferencevaluespreviouslydeterminedinhealthy children(37.2±5.7pg/ml)[16]andshowedsignificantlylower lev-els(p=0.003)inobesechildrenwithrespecttocontrols,mimicking thetrend alreadyobserved in obeseadults(Fig.1b). Whenthe resultswereanalyzedasafunctionofgender,nosignificant dif-ferenceforCNPvalueswasfoundbetweenmalesandfemales.A significantpositivecorrelationbetweenBNPandglycemia(r=0.33; p<0.05)wasobserved.

Consideringthebiochemicalparametersanalyzedinobese chil-dren(glycemia,insulin, cholesterol,HDL, LDLand triglycerides) theonlysignificantcorrelationsresultedbetweenCNPand HDL (r=0.37, p=0.048) as well as between BNP and HDL (r=0.4, p=0.029)andtriglycerides(r=0.5,p=0.005)confirmingtheirrole inlipolysisandadipogenesis[3,32].

NosignificantcorrelationwereobservedbetweenCNPorBNP andthemorphometricfeatures.

4. Discussion

ThisstudyreportsforthefirsttimethebehaviorofCNPplasma levelsinobeseadolescents,showingthatoverweightandobese childrenhavesignificantlylowerlevelsthannormal-weight sub-jects. BNP plasma levels measured in the same subjects also resultedlower,confirmingreportsfrompreviousinvestigatorsin obeseadults[21,28,29,32,47]andsuggestingadefectivenatriuretic peptidesysteminthesepatients.

Thereareseveralmechanismsthatcouldbepotentially respon-siblefortheinverseassociationbetweennatriureticpeptideplasma levelsandbodymassindex[2,24].Oneofthesemaybeanincreased expressionofthenatriureticpeptideclearancereceptors(NPR-C) onadipocytecellsand/orimpairedsynthesis/releaseofnatriuretic peptidesfrommyocytesfoundinobesesubjects[1].Another mech-anismforthereducedsecretionofnatriureticpeptidesmightbean impairedmyocardialhormonerelease[23]orsynthesis[31]asa consequenceofoverweight/obesity.Recently,weobservedlower levelsofBNP,ANPandCNPmRNAexpressionincardiactissueof obeseratsincomparisonwiththeirnormal-weightcounterparts, suggestingthatobesitymaybeassociatedwithadecreased syn-thesisofthesepeptidesbycardiaccells[3].Thus,wehypothesize thatasimilarobesity-inducedmechanismmightalsobeoperating inobesehumans,includingchildrenandadolescents.

Thus,inouroverweight/obeseadolescentsadecreased synthe-sisandincreasedclearancemayworksynergisticallytolowerthe circulatinglevelsofnatriureticpeptides.

0 2 4 6 8 10 12 14 16 18

CN

P

concentration

(p

g/ml)

HEALTH

Y

ADOLESCENTS

ADOLESCENTS

OBESE

*

BN

Pc

oncentration ( pg/ml)

HEALTH

Y

ADOLESCENTS

ADOLESCENTS

OBESE

#

Alternatively,ithasbeenhypothesizedthatthedecreased lev-elsof circulatingnatriureticpeptidearenottheconsequenceof overweight/obesity,butratheracausativefactorinthegenotype orphenotypeleadingtodevelopmentofobesity[21].Infact,both ANPandBNParenowrecognizedfactorsinvolvedinfatmetabolism asstimulatorsoflipolysisinadiposetissue[19].

Data on plasma natriuretic peptide levelsin obese children andadolescentsarescarce,limitedtopro-BNPandnotunivocal. To our knowledge there are only two studies in obese chil-dren/adolescents,showingcontrastingresults.Onestudyreported higherlevelsofpro-BNPinobesevsnormal-weightcontrols[41], whiletheotherstudydidnotfindanydifferencebetweenobese andnon-obesesubjects[42].Bothstudiessuggest that circulat-ing pro-BNP in obese children/adolescents behaves differently than in obese adults, who have reduced levels of the peptide [21,28,29,32,47].Whileourresultsagreewiththosefoundinobese adults,theydonotagreewithdatareportedinchildren/adolescents [41,42].Wedonothaveaclear-cutexplanationforthedifferent resultsincirculatingBNPlevelsfoundamongourownandothers’ studies.Onepossibilitymightbethedifferentobesepopulations examined,whichwereyoungerthanours,spanningfromaboutage 5to14years[41]andfromage5to17years[42],andthedifferent assaymethodused.ItisknownthatBNPandNT-proBNPresults dependontheassaymethodusedandontheageandgenderofthe populationstudied[4].

In recent years, the childhood obesity epidemic has begun to compromise the health of the pediatric population by pro-motingprematuredevelopmentofatherosclerosisandmetabolic syndrome,bothofwhichsignificantlyincreasetheriskof cardio-vasculardiseaseearlyinlife.Giventheinvolvementofnatriuretic peptidesintheseconditions[17,25],itbecomesextremely impor-tanttohave age-specificreferenceintervalsfor thispeptide,as reportedinsomerecentlypublishedstudiesonnatriureticpeptides [4,16,27].

Alimitationofthisstudyisthelackofcontrolsubjects.However, weusedourownreferencedata,specificfortheBNPandCNPassay methodsemployedinthisstudy.Itisalsointerestingtonotethat bothBNPandCNPhadthesamepatterninouroverweight/obese subjects,supportingthatanalteredregulationofnatriuretic pep-tidesmaybeworkingatthisage,aspreviouslyobservedinadults. ThesedatasuggestthatCNPdetermination,alongwiththatofthe othernatriureticpeptides,maybeconsideredacomplementary toolforthecharacterizationofthesesubjects.Inthisregard,the availabilityofdrugspromotingtheactionsofnatriureticpeptides maybeanattractivetherapeuticoptionforpreventing cardiovas-culardisease.

Funding

Thisstudywasconductedwithinthecontextoftheproject enti-tled“Earlydiagnosisoforganmetabolicandinflammatorydamage related with cancer and cardio-metabolic risk in childhood obe-sity.Validation ofpanel-orientedbiomarkersinobeseanimalsand implementationinchildrenandadolescents”(UniqueProjectCode B55E09000560002),supportedbytheRegioneToscana(Tuscany Region)undertheResearchCall“InnovationinMedicine2009”.

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