j our na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / d r u g a l c d e p
Shortcommunication
High-sensitivitygamma-glutamyltransferasefractionpattern
inalcohol
addicts
andabstainers
MariaFranzinia,c,∗,IreneFornaciaria,TizianaVicob,MarcoMoncinib,ValerioCellesib,
MiloMeinib,MicheleEmdinc,AldoPaolicchic,d
aScuolaSuperioreSant’Anna,Pz.MartiridellaLibertà33,56127Pisa,Italy
bDrugAddictionDepartment,AziendaASL5,ViaFleming1,56025Pontedera(Pi),Italy
cDivisionofCardiovascularMedicine,FondazioneG.MonasterioCNR–RegioneToscana,ViaGiuseppeMoruzzi1,56124Pisa,Italy dDepartmentofExperimentalPathology,UniversityofPisa,ViaRoma55,56126Pisa,Italy
a r t i c l e i n f o
Articlehistory:
Received22December2011 Receivedinrevisedform12April2012 Accepted4June2012
Available online 29 June 2012
Keywords: Alcohol Biomarker
Gamma-glutamyltransferasefractions Gelfiltrationchromatography
a b s t r a c t
Background:Fourfractionsofgamma-glutamyltransferase(GGT)withdifferentmolecularweight(b-,m-,
s-,andf-GGT)arepresentinhumanplasma.DifferentialGGTfractionpatternisfoundinnon-alcoholic
liverdisease(NAFLD)andchronicviralhepatitis,characterizedbynormalordecreasedb-GGT/s-GGT
(b/s)ratio,respectively.
Methods:ChromatographicfractionalGGTanalysiswasperformedonplasmaobtainedfrom51subjects:
27alcoholics(mean(SD),age45(9)years;23males;14positiveforviralinfection),24abstinentsfrom
atleast1month(43(12)years;20males;6positiveforviralinfection).Twenty-sevenblooddonors
matchedforageandgender(44(9)years;23males)wereselectedascontrols.
Results:Allfractionsweresignificantlyincreasedinalcoholics(P<0.001),s-GGTshowingthelargest
increase,whileonlym-GGTands-GGTwereelevatedinabstainers(P<0.01),incomparisonwithcontrols.
Theb/sratiowassignificantlylowerinbothalcoholicsandabstainersthanincontrols(median(25th–75th
perc.):0.10(0.07–0.15),0.16(0.10–0.24),0.35(0.29–0.53),respectively,P<0.001).Viralinfectiondidnot
significantlychangesabsolutevaluesofindividualGGTfractionsinalcoholics,buttheb/sratiowas
significantlylowerinviruspositivethaninvirusnegativesubjects(0.08(0.05–0.12),0.14(0.09–0.20),
respectively,P<0.01).
Conclusions:ThefractionpatternanalysismightincreasethespecificityofGGTasbiomarkerofalcohol
abuse,especiallyconcerningthedifferentialdiagnosisbetweenalcoholismandNAFLD,acommoncause
ofelevatedGGTlevelinthegeneralpopulation.
© 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Serumgamma-glutamyltransferase(GGT)isawell-recognized sensitivebiomarkerofalcoholabuse(RosalkiandRau,1972;Das etal.,2008).GGTspecificityanddiagnosticaccuracyforalcoholism arereducedbythefactthatserumGGTelevatedlevelsareencoun- teredinvariousphysiologicalandpathologicalsettingincluding awidearrayofhepatobiliarydisorders(DhanyaandVasudevan, 2008).Inparticular,acommoncauseofserumGGTincreaseisthe non-alcoholicfattyliverdisease(NAFLD),thataffectsa growing proportionofthepopulation(Bedognietal.,2005;Tahanetal.,
∗ Correspondingauthorat:ScuolaSuperioreSant’Anna,c/oFondazioneToscana G.Monasterio,ViaG.Moruzzi1,56124Pisa,Italy.Tel.:+390503153309; fax:+390503152166.
E-mailaddresses:m.franzini@sssup.it,franzinimaria@gmail.com(M.Franzini).
2008),thusthepossibilityofdiscriminatingbetweenthetwodis- easeswouldhelpthediagnosisofbothconditions.
Total GGT activity corresponds to several distinct GGT- containingmolecularcomplexes,withdifferentphysico-chemical properties(Huseby,1982),pathophysiologicalandclinicalcorre- lations(NemesanszkyandLott,1985).Recently,ahighsensitivity clinicallaboratorymethodhasbecomeavailable,thatallows,in humanplasma,thesimultaneousdetectionoffourdifferentfrac- tionsof GGT, namelyb-GGT,m-GGT,s-GGT, and f-GGT,having molecularweight rangingfrom 2000to70kDa (Franzini etal., 2008a).Whilef-GGTisthemostabundantGGTfractioninhealthy individuals(Franzinietal.,2008b),theotherfractionsareresponsi- bleforGGTincreaseindiseasedones.Recently,wefounddifferent GGTfractionpatternscharacterizingeitherNAFLD,orchronicviral hepatitisC(CHC):theformerwascharacterizedbyanincreasein allfractionswithoutchangingintheb-GGT/s-GGT(b/s)ratio,and thelaterbyaprominentincreaseins-GGTtogetherwithamodest
Controls(n=27) Alcoholics(n=27) Abstainers(n=24) P Controlsvs. alcoholics P Controlsvs. abstainers P Alcoholicsvs. abstainers Males,n 23 23 20 Age,years 44.0(35.0–48.0) 44.0(36.0–48.0) 39.0(35.5–53.0) n.s. n.s. n.s. BMI,kg/m2 24.8(22.9–27.0) 23.5(21.6–27.3) 24.3(22.0–26.1) n.s. n.s. n.s. AST,U/L 20.0(17.0–23.0) 58.0(31.0–96.0) 22.5(18.0–33.0) <0.001 n.s. <0.001 ALT,U/L 24.0(15.0–26.0) 57.0(25.0–95.0) 29.0(15.0–41.5) <0.01 n.s. <0.05 MCV,fL 85.9(83.3–87.5) 95.1(89.2–102.3) 93.1(91.0–95.3) <0.001 <0.001 n.s. Bilirubin,mg/dL 0.80(0.60–0.90) 0.76(0.45–1.11) 0.44(0.32–0.81) n.s. n.s. n.s. TotalGGT,U/La 23.9(15.1–32.0) 133.0(50.1–287.0) 37.5(19.4–84.8) <0.001 <0.05 <0.001 b-GGT,U/La 2.4(1.4–4.5) 7.5(3.2–21.0) 2.8(1.3–7.6) <0.001 n.s. <0.01 m-GGT,U/La 1.0(0.5–1.5) 5.2(2.8–19.0) 2.1(0.8–4.5) <0.001 <0.01 <0.001 s-GGT,U/La 7.3(3.6–12.4) 95.7(30.1–207.8) 15.3(8.1–58.5) <0.001 <0.01 <0.001 f-GGT,U/L 13.7(9.9–16.9) 22.7(15.4–26.7) 14.4(9.0–18.8) <0.001 n.s. <0.001 b/sratioa 0.35(0.29–0.53) 0.10(0.07–0.15) 0.16(0.10–0.24) <0.001 <0.001 <0.05
Dataarepresentedasmedian(25th–75thpercentile).BMI:bodymassindex;AST:aspartate-aminotransferase;ALT:alanineaminotransferase;MCV:meancorpuscular volume;GGT:gamma-glutamyltransferase.Statisticalanalysis:1-wayANOVAfollowedbyTukey’smultiplecomparisontest.n.s.:notsignificant.
aStatisticalanalysisperformedonln-transformeddata.
increaseinotherfractionsanddecreaseoftheb/sratio(Franzini etal.,2011).
Sincesteatosisisamainfeatureofhepatocellulardamageoccur- ringearlyinthecourseofalcoholicliverdisease(ALD;Basraand Anand,2011),weaimedtocheckwhethertheGGTfractionpattern associatedwithALDcouldmimicthosefoundinNAFLD(Franzini etal.,2011),inapreliminaryseriesofalcoholaddicts.Thiswould permittodesignlarge-scalestudiesconcerningthediagnosticvalue ofGGTfractionanalysisinthesettingofalcoholabuse.
2. Methods
2.1. Subjects
Fastingbloodsampleswereobtainedfrom51subjectswithpositivehistoryof alcoholdependenceaccordingtotheICD-9-CMDiagnosisCode303.9(mean(SD; range):age43.6(10.6;26–69)years;45malesand6females;abuseduration4.6 (range1.3–20)years).AllthesesubjectswereenrolledinthestudyattheAlcoholism CenteroftheDrugAddictionDepartmentoftheLocalHealthService(Pisa,Italy).
Alcoholdependenceandconsumptionwereestimatedbyphysiciansofthe AlcoholismCenter baseduponthe informationcollected from thepatientas well asfrom his/her relatives during the interview.The alcohol content of onestandarddrinkinItaly is assumedto be12g ofpurealcohol (website:
http://icap.org/PolicyIssues/DrinkingGuidelines).AccordingtoWHOrecommenda- tionswesetthelimitofhazardousalcoholconsumptionto>45g/dayformenand >30g/dayforwomen(Schellenbergetal.,2005).
Twenty-foursubjectswereabstinentforatleast1month(age43.3(12.1;26–69) years;20men;abstinencetime:2.7(3.4)months):thesesubjectswillbereferredas “abstainers”.SixabstainerswereeitherhepatitisCvirus(HCV)orhepatitisBvirus (HBV)antibodiespositive.Abnormalitiesinbothalanine(ALT)andaspartate-amino transferase(AST)havebeendetectedin3abstainers.Theother3abstainersshowed ALTand/orASTelevation.
Twenty-sevensubjectswerecurrentlyalcoholdependent(age44.9(9.2;30–65) years;23men;alcoholconsumption:209(192;100–810)g/day).Fourteensubjects (age40.1(6.5;30–50)years;12men;alcoholconsumption162(78;80–290)g/day) wereHCVorHBVantibodiespositivewithbothASTandALTelevatedlevels,10 amongthemwereprovedpositiveforHCV-RNAtestorHBVsurfaceantigen(HBsAg). Theremaining13subjects(age48.0(10.1;33–65)years;11men;alcoholconsump- tion250(254;100–810)g/day)showednegativevirologyanalysis,amongthem,7 showedASTandALTelevation.
Twenty-sevenblooddonorsmatchedforageandgenderhavebeenselectedas controls(age43.7(9.3;27–64)years;23men;alcoholconsumption≤20g/day).
TheInstitutionalEthicsCommitteeapprovedthestudyandallsubjectsgave informedconsent.
2.2. Laboratoryanalyses
Fastingbloodsamples,collectedinethylenediamine-tetra-aceticacid(EDTA), wereobtainedfromallsubjects.AST,ALT,meancorpuscularvolume(MCV),total bilirubin,serumglucose,totalcholesterol,HDLcholesterol,LDLcholesterol,triglyc- eridesandcreatinineclearance(eGFR)wereassayedwithin3haccordingtothe standardclinicallaboratoryproceduresbyautomatedanalyzers(BeckmanSynchron CX9-PROanalyzer,AbbottCell-DynSaphireforbloodcellcount).LDLcholesterol
andeGFRwerecalculatedusingtheFriedewaldandtheCockcroft-Gaultformula, respectively.
2.3. FractionalGGTanalysis
AliquotsofplasmaEDTAsampleswerestoredat−20◦Candusedwithinthree
monthsforfractionalGGTanalysis.AnalysisoftotalandfractionalGGTwasper- formed,aspreviouslydescribed(Franzinietal.,2008a,b),usingafastprotein liquidchromatographysystem(AKTApurifier;GEHealthcareEurope,Milan,Italy) equippedwithagel-filtrationcolumn(Superose6HR10/300GL;GEHealthcare Europe)andafluorescencedetector(JascoFP-2020;JascoEurope,Lecco,Italy). Theenzymaticactivitywasquantifiedbypost-columninjectionofthefluores- centsubstrateforGGT,gamma-glutamyl-7-amido-4-methylcoumarin(gGluAMC). Enzymaticreaction,inthepresenceofgGluAMC0.030mmol/Landglycylglycine 4.5mmol/L,proceededfor4.5mininareactioncoil(PFA,2.6mL)keptatthe37◦C
inawaterbath.Thefluorescencedetectoroperatingatexcitation/emissionwave- lengthsof380/440nmdetectedtheAMCsignal.Areaundercurvechromatogram curveisproportionaltoGGTactivity,whichwasquantifiedaspreviouslydescribed (Franzinietal.,2008b).
2.4. Statisticalanalysis
Statisticalanalysiswasconductedbyone-wayANOVAanalysisfollowedby Tukey’smultiplecomparisontestorStudent’sttest.Total,b-,m-ands-GGT,aswell asb/sratioandtriglyceridevalueswereln-transformedtoreducethedistribution skewness.
3. Results
As expected, significant elevation of aspartate-amino trans- ferase(AST, P<0.001),alanine-amino transferase(ALT,P<0.01), andmean corpuscularvolume(MCV,P<0.001)wasdetectedin alcohol addicts ascompared to controls.Abstainers showedan increaseinMCVvaluesonly(P<0.001;Table1).
AlthoughtotalGGTvaluesweresignificantlyincreasedinalco- holaddictsandabstainersascomparedtocontrols(P<0.001and <0.05, respectively), while all GGT fractions were significantly higherinalcoholics(P<0.001),onlym-GGTands-GGTremained significantlyelevatedin abstainers(P<0.01;Table1).In alcohol addicts,a13.1-foldincreasevalueins-GGTwasdetected,together withasignificantbutmoremodestelevationinallotherfractions (f-GGT:1.7-fold;b-GGT:3.1-fold;m-GGT:5.2-fold).Inabstainers, asstated above,thevalues ofboth m-GGTand s-GGTfractions remainedsignificantlyhigherthanincontrols,butatamuchlower extent(2.1-foldfor both).Thustheb/s ratioremainedlowerin bothgroups(P<0.001),thanincontrols,withahigherb/sratio inabstainersthaninaddicts(P<0.05;Table1).
Amongaddicts,nosignificantdifferencesasconcernseachGGT fractionwere found between theHCV/HBV positive or (n=14) or negative (n=13) subjects, except for the b/s ratio,that was
Alcoholics HCV/HBVnegative(n=13) HCV/HBVpositive(n=14) P TotalGGTa 168.0(45.7–695.4) 129.0(73.2–274.5) n.s. b-GGTa 9.0(3.0–60.0) 5.6(3.4–14.4) n.s. m-GGTa 8.4(2.5–32.2) 4.5(2.9–15.3) n.s. s-GGTa 112.9(24.6–502.1) 95.6(48.8–205.3) n.s. f-GGT 22.7(14.0–55.3) 22.7(18.0–25.5) n.s. b/sratioa 0.14(0.09–0.20) 0.08(0.05–0.12) 0.0045
Abstainers HCV/HBVnegative(n=18) HCV/HBVpositive(n=6) P
TotalGGTa 37.5(18.0–261.8) 47.1(25.0–130.3) n.s. b-GGTa 2.7(1.1–25.6) 3.0(1.5–6.9) n.s. m-GGTa 2.0(0.7–4.6) 2.1(1.1–6.7) n.s. s-GGTa 15.3(7.1–56.7) 30.1(9.2–94.8) n.s. f-GGT 14.2(8.2–19.0) 14.4(10.9–21.1) n.s. b/sratioa 0.17(0.11–0.25) 0.11(0.05–0.25) n.s.
Dataarereportedasmedian(25th–75thpercentile).
aStudent’sttesthasbeenperformedonln-transformeddata.
significantlyhigherinthelattergroup(P<0.01;Table2), butin bothcasessignificantlylowerthanincontrols(P<0.001forboth comparisons).Withinabstainersnodifferenceswerefoundinb/s ratio,betweenHCV/HBVpositiveornegativesubjects(Table2).
4. Discussion
InthepresentstudyweshowthattheelevationoftotalGGTin alcoholaddictsisassociatedwithaprominents-GGTincrease,and alesserincreaseofallthreeotherfractions.Forthisreason,theb/s ratioresultedsignificantlylowerthanincontrols.
ThisGGTfractionpatterncorrespondstothatfoundinsubjects withchronicviralhepatitisC(CHC),butnotinsubjectswithnon- alcoholicfattyliverdisease(NAFLD)whoarecharacterizedbyb/s ratiovaluescomparabletothoseofhealthysubjects(Franzinietal., 2011).Interestingly,despitesimilarabsolutevaluesoftotalGGT anditsfractions, thedecreaseinb/sratio wasmore markedin alcoholaddictsprovedtobepositiveforviralinfection,suggesting thatb/sratioisadistinctandpotentiallyquantitativebiomarkerof hepatocellulardamageinalcoholism,eveninthepresenceofviral infection.
AbstinencefromalcoholresultedinloweringoftotalGGTval- ues:b-GGTandf-GGTfractionsreturnedtonormalvalues,while m-GGTands-GGTlevelsremainedpersistentlyhigh,inadditionto alowerb/sratiothanincontrols.Thisfindingconfirmsb/sratioasa sensitivebiomarkerofpersistentliverdamage,independentlyfrom totalGGTlevel.Inaddition,thedifferentialdecreaseintheGGT fractionsinalcoholabstainerssuggeststhatGGTfractionanalysis mightperformbetterthantotalGGT,asformonitoringabstinence. Concerningtheinterferencebetweenviralhepatitisandalcohol addiction,theb/sratioshowedasignificantdifferencebetween HCV/HBV positive and negativealcohol addicts,but not among HCV/HBVpositiveandnegativeabstainers;anywayitislikelytobe duetothemodestsamplesize(6positivevs.18negativeabstain- ers);largerstudiesareneededtofullyexplorethediagnosticvalue oftheb/sratiointhisfield.
Ina previousstudy,weshowedthat fractionalGGTanalysis showedhigherspecificityand sensitivityfor thedirectand dif- ferentialdiagnosisofNAFLDandCHCincomparisontototalGGT activity(Franzinietal.,2011).WhenconsideringthatamongNAFLD subjects,highserumGGTlevelshavebeenalreadyidentifiedaspre- dictorsofliverfibrosisrisk(Tahanetal.,2008),furtherandlarger prospectivestudiesareneededtoestablishthepotentialvalueof theindividualGGTfractionsandoftheb/sratio,eitherinthescreen- ingofalcohol abuse,oraspredictorsofchronicliverdamagein
Roleoffundingsource
ThisworkwassupportedbyInstitutionalfunding(G.Monas- terio Foundation CNR-Regione Toscana, Scuola Sant’Anna and UniversityofPisa,Italy).Allfundingbodieshadnofurtherrolein studydesign;inthecollection,analysisandinterpretationofdata; inthewritingofthereport;orinthedecisiontosubmitthepaper forpublication.
Contributors
AuthorsAP,MEandMMdesignedthestudyandwrotethepro- tocol.AuthorTV,MMandVCselectedparticipantstothestudyand providedplasmasamples.AuthorIFperformedfractionalGGTanal- ysis.AuthorMFundertookthestatisticalanalysiswithsignificant scientificinputfromMEandAP.AuthorMFwrotethefirstdraft ofthemanuscriptandauthorsAP,MEandMMcontributedtothe maincontentandprovidedcriticalcommentsonthefinaldraft.All authorsapprovedthefinalmanuscript.
Conflictofinterest
Noconflictdeclared.
Acknowledgements
Theauthorsthankthestudyparticipantsfortheircontribution totheresearch.
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C
HAPTER5
C
IRCULATING GAMMA-
GLUTAMYLTRANSFERASEFRACTIONS IN LIVER CIRRHOSIS
C I R R H O S I S A N D L I V E R F A I L U R E