Amylase(serum,plasma)

1.1

1.2

1.3

1.4

1.4

Nameanddescriptionofanalyte
Nameofanalyte
Amylase(serum,see10.2)
Alternativenames
Amylase,1,4DGlucanglucanohydrolase(EC3.2.1.1)
Abbreviation:AMY
Isoenzymes:AMY1(ptyalin,Stype,SAMY);AMY2(Ptype,PAMY)

NLMCcode

Descriptionofanalyte
AmylaseisaheterogeneouscalciumdependentmetalloenzymeofMW
5462kDa.Itexistsastwoisoenzymes:pancreatic(Ptype)andnon
pancreatic(Stype).Theseisoenzymesareproductsoftwocloselylinked
locionchromosome1.Additionalamylaseheterogeneityisduetoallelic
variation(Stype,12alleles;Ptype,6alleles).Bothalsoundergopost
translationalmodificationbydeamidation,glycosylation,and
deglycosylationtoformvariousisoforms.
AmylasehasawidetissuedistributionwiththehighestactivitiesoftheP
andStypesbeingfoundintheexocrinepancreasandsalivaryglands,
respectively.Ptypeamylaseissynthesizedbypancreaticacinarcellsand
secretedintotheintestinaltractviathepancreaticductsystem.Itsaction
isfavouredbythemildlyalkalineconditionsintheduodenum.The
greatestStypeamylaseactivityisinsalivaryglands,whereitinitiatesthe
hydrolysisofstarcheswhilefoodisinthemouthandoesophagus.Its
actionisterminatedbyacidinthestomach.Stypeamylaseisalsofoundin
extractsoftestes,ovaries,fallopiantubes,mullerianducts,striatedmuscle,
lungsandadiposetissue,aswellasinsemen,colostrum,tearsandmilk.
About25%ofplasmaamylaseisexcretedbythekidneysbutthe
majorityisreabsorbedintheproximaltubules.Theliverissuspectedto
bethemajororganforamylaseremoval,resultinginahalflifeofabout
10h.

Functionofanalyte
Amylaseisanendoglycosidaseenzymeofthehydrolaseclassthat
catalysesthehydrolysisof1,4glucosidiclinkagesbetweenadjacent
glucoseunitsincomplexcarbohydrates.Bothstraightchain(linear)
polyglucans,suchasamylose,andbranchedpolyglucans,suchas
amylopectinandglycogen,arehydrolysed,butatdifferentrates.Inthe
caseofamylose,theenzymesplitsthechainsatalternate1,4
hemiacetal(COC)links,formingmaltoseandsomeresidualglucose.In
thecaseofbranchedpolyglucans,maltose,glucoseandaresidueoflimit
dextrinsareformed.Theenzymedoesnotattackthe1,6linkagesatthe
branchpoints.
Thefunctionalintegrityofamylaseisabsolutelydependentonthe
presenceofcalcium.However,fullintegrityisdisplayedonlyinthe
presenceofanyoneofvariousanions,includingchloride,bromide,

Copyright Association for Clinical Biochemistry 2012

2.1

2.2

3.1

3.2

4.1

nitrate,cholateormonohydrogenphosphate.Chlorideandbromideare
themosteffectiveactivators.ThepHoptimumis6.9to7.0.

Samplerequirementsandprecautions
Mediuminwhichmeasured
Amylaseismainlymeasuredinserum.Measurementsinurineare
sometimesvaluable.
Amylaseisoccasionallymeasuredinascitic,peritonealorpleuralfluid
whereitspresencecanindicatepancreatitisorthepresenceofatumour.

Precautionsresampling,handlingetc.
Withtheexceptionofheparin,allcommonanticoagulantsinhibitamylase
activitybecausetheychelatecalcium:citrate,ethylenediaminetetraacetic
acidandoxalateinhibititbyasmuchas15%.Therefore,amylaseassays
shouldbeperformedonlyonserumorheparinisedplasma.
Amylaseisstable:activityisfullyretainedduringstoragefor4daysat
roomtemperature,2weeksat+4C,1yearat25C,and5yearsat75
C.
Summaryofclinicalusesandlimitationsofmeasurements
Uses
1.ThemainapplicationformeasuringtotalamylaseorPtypeamylaseis
tosupportadiagnosisofacutepancreatitis:serumamylaseactivity
typicallyincreaseswithin212hofonsetofsymptoms,hasa24hpeak
(oftenmorethanfivetimestheupperlimitofnormal)andremains
elevatedfor37days.
2.Amylaseisoenzymefractionationmaybevaluableindetermining
whetherhyperamylasaemia(see6.2)inHIVpatientsreceivinghighly
activeantiretroviraltherapyincludingnucleosideanalogues(ddI,ddC
etc)isduetodruginducedpancreatitisoranincreaseinthenon
pancreaticfraction.
Limitations
AsignificantproportionofsubjectsofAfricanandAsianoriginhaveanS
typeamylaseactivityabovethereferenceintervalderivedfromCaucasian
populations.Thiscanresultinanapparentlyelevatedtotalamylasethatis
nonpathological.Thepresenceofmacroamylasaemia(see6.2)mayalso
resultinanonpathologicalincreaseintotalamylaseactivity.
Measurementoftotalserumamylaselacksspecificityforacute
pancreatitissinceitcanalsoberaisedinappendicitis,renaldisease,
pregnancy,diabeticketoacidosisandgastrointestinaldisease,among
manyothercauses(see6.2).

Analyticalconsiderations
Analyticalmethods
A.Totalamylase
Therearemoreapproachestomeasuringamylasethanalmostanyother
commonclinicalanalyte.Historically,saccharogenic,amyloclasticand
chromolyticstarchmethodsweretheassaysofchoice,butthesearenow
obsolete.Theyhavebeenreplacedbymethodswithwelldefined

Copyright Association for Clinical Biochemistry 2012

substrates.This,andtheuseofauxiliaryandindicatorenzymes,has
improvedthereactionstoichiometryandledtomorecontrolledand
consistenthydrolysisconditions.Substratesusedincludesmall
oligosaccharidesand4nitrophenyl(4NP)glycosidesubstrates.
1. Glucosidase(maltase:E.C.3.2.1.20)usedincombinationwith
hexokinase(E.C.2.7.1.1)coupledwithglucose6phosphate
dehydrogenase(Dglucose6phosphate:NAD(P)+1oxidoreductase,
EC.1.1.1.49).Thereactionsare
a. Maltopentose(amylase)maltotriose+maltose
b. Maltotriose+maltose(glucosidase)5glucose
c. Glucose+ATP(HK)glucose6phosphate(G6P)+ADP
d. G6P+NAD+(G6Pdehydrogenase)6phosphogluconolactone
+NADH
TheformationofNADHismeasuredspectrophotometricallyat340
nm.
2. Maltosephosphorylase(maltose:phosphate1Dglucosyltransferase:
E.C.2.4.1.8)usedincombinationwithphosphoglucomutase(D
glucose1,6phosphomutase,EC.5.4.2.6)coupledwithglucose6
phosphatedehydrogenase(Dglucose6phosphate:NAD(P) +1
oxidoreductase,EC.1.1.1.49)toavoidinterferencefromendogenous
glucose.Thereactionsare:
a. Maltotetraose(amylase)2maltose
b. Maltose+inorganicphosphate(maltosephosphorylase)
glucose+glucose1phosphate(G1P)
c. G1P(phosphoglucomutase)G6P
d. G6P+NAD+(G6Pdehydrogenase)6phosphogluconolactone
+NADH
Foreachbondhydrolysedbyamylase,twomoleculesofNADHare
produced,butonlyifneitherglucosenormaltotrioseisproducedby
theactionofamylase.Inpractice,theseproductsarepresentin
significantquantitiesinthereactionmixture;therefore,thismethod
underestimatesamylase.
3. Glucosidase(maltase:E.C.3.2.1.20)incombinationwith4
nitrophenolglycosidesubstrates.Thereactionsare:
a. 4NP(glucose)7(amylase)4NP(glucose)4,3,2
b. 4NP(glucose)4,3,2(glucosidase)4NP(glucose)4+glucose
+4NP
Theresultofthecombinedhydrolysisbyamylaseinthespecimenand
bythereagentglucosidaseisthat>30%oftheproductisfree4NP.
Theformationoffree4NPismeasuredspectrophotometricallyat405
nm.
Glucosidasedoesnotreactwitholigosaccharidescontainingmore
thanfourglucosemolecules.Originally,therewereproblemswiththe
poorstabilityofthereconstitutedassaymixturebecauseoftheslow
hydrolysisofthe4NPglycosidebyglucosidase.Thishasbeen
reducedbycovalentlylinkingablockinggroup,e.g.a4,6ethylidene
group,tothenonreducingendofthemolecule(EPS,ethylidene
protectedsubstrate).Theblockedsubstratealsoshowsadifferentand
moreadvantageoushydrolysispattern.Theethylidene4NP
(glucose)7substratefragmentsapproximatelyas4NP(glucose)2
(40%),4NP(glucose)3(40%)and4NP(glucose)4(20%).
4. Glucosidase(maltase:E.C.3.2.1.20)incombinationwithblocked4
nitrophenolglycosidesubstrates.Thereactionsare:
Copyright Association for Clinical Biochemistry 2012

4.2

4.3

a. EPS4NP(glucose)7+H2O(amylase)EPS4NP(glucose)x
+4NP(glucose)7x
b. 4NP(glucose)7x+(7x)H2O(glucosidase)4NP+(7x)
glucose
IFCChasoptimisedthismethodat37C,andthisisthereference
procedure.
5. Analternativemethodbasedonthe2chloropnitrophenol(CNP)
indicatoruses2chloropnitrophenylDmaltotrioside(CNPG3)asa
substrate.Thereactionis:
10CNPG3(amylase)9CNP+CNPG2+9maltotriose+
glucose
Thisassaydoesnotrequireglucosidasesandisconsideredadirect
assay.Itsdisadvantagesincludelowsubstrateconversionrateandthe
variationinmolarabsorptivityofCNPassociatedwithchangesinpH,
temperatureandproteincontent,aswellasthepresenceofthe
activator,potassiumthiocyanate,causingallostericchangesto
amylaseandprecludingtheuseofantibodiesforPtypeamylase
determination.

B.Amylaseisoenzymedifferentiation
PtypeamylasecanbedifferentiatedfromtheStypeamylasebyselective
inhibitionofStypebyawheatgerminhibitor,temperatureinhibition,
immunoprecipitationorimmunoinhibitionbyamonoclonalantibody.
However,onlythemethodsbasedonselectiveinhibitionbymonoclonal
antibodieshaveshownsufficientprecision,reliability,practicability,and
analyticalspeedtoallowreliablemeasurementofPtypeamylase.A
commerciallyavailableassaythatusesthesynergisticactionoftwo
immunoinhibitorymonoclonalantibodiestoStypeamylaseisavailable.
TheuninhibitedPtypeamylaseactivityismeasuredusingEPS4NP
(glucose)7asasubstrate.FalsepositivePtypeamylaseresultshavebeen
reportedinsubjectswithmacroamylasaemia,asthecomplexdiminishes
theabilityofmonoclonalantibodiestoefficientlyinhibitStypeamylase.

C.Amylaseisoformseparation:
Theamylaseisoformscanbeseparatedbyisoelectricfocusing,ion
exchangechromatographyorgel/capillaryelectrophoresisby
electrophoreticendosmosis.

D.Determinationoftheamylasemacrocomplex:
1. Electrophoresis:macroamylaseformsabroadmigratingband
differentfromthehomogeneousbandsproducedbytheamylase
isoenzymespresentinserum.
2. Precipitation:usingapolyethyleneglycol(PEG)6000solution(240
g/L);residualamylaseactivityof<30%inthesupernatantis
indicativeofsignificantmacroamylase.

Referencemethod
Thereferencemethodisanoptimisedversion(at37oC)ofthemethod
shownin4.1(4).

Referencematerials
Primaryreferencematerial:amylasefromhumanpancreas
(IRMM/IFCC456).

Copyright Association for Clinical Biochemistry 2012

4.4

4.5

Interferingsubstances
Highserum[triglyceride]interfereswithsomeamylaseassays.
Sourcesoferror
Amylaseisinhibitedbymostnonheparinanticoagulantsincluding
citrate,ethylenediaminetetraaceticacidandoxalate.

Referenceintervalsandvariance

5.1.1 Referenceinterval(adults)
Serumamylaseismethoddependent.UsingtheIFCCrecommended
methodat37oC,theserumreferenceintervalinadultsis28100U/L
(0.481.70kat/L).Ptypeamylaserepresents3550%ofthenormal
serumamylase,withStypeamylaseformingtheremainder.
5.1.2 Referenceintervals(others)
Afterbirth,Stypeamylaseactivityincreasesinserumsteadilywithage
andreachesnormaladultvaluesat2yearsofage.Ptypeamylaseisnot
demonstrableinseruminmostchildrenlessthan6monthsold,but
activityrisesslowlythereaftertoreachadultlevelsat5yearsofage,
reflectingpostnataldevelopmentofexocrinepancreaticfunction.
Thereferenceintervaldoesnotdifferbetweenmalesandfemales.
5.1.3 Extentofvariation
5.1.3.1 InterindividualCV:32.1%
5.1.3.2 IntraindividualCV:10.8%
5.1.3.3 Indexofindividuality:0.34
5.1.3.4 CVofmethod:typically<3%
5.1.3.5 Criticaldifference:30.3%
5.1.4 Sourcesofvariation
TheStypeamylasegenehasundergoneduplicationduringevolution,and
manyindividualshavemultipletandemrepeatsofthegene.Thegene
copynumberisassociatedwithapparentevolutionaryexposuretohigh
starchdiets.SubjectsofAfricanandAsianorigintendtohaveahigher
totalamylaseactivity(duetoincreasedStypeamylaseactivity)
comparedwiththoseofCaucasiandescent.

6
Clinicalusesofmeasurementandinterpretationofresults

6.1 Usesandinterpretation
Diagnosisofacutepancreatitis
Adiagnosisofacutepancreatitisrequirestwoofthefollowingthree
features:abdominalpaincharacteristicofacutepancreatitis;serum
amylase(and/orlipase)3timestheupperlimitofnormal(ULN),and
characteristicfindingsofacutepancreatitisonCTscan,MRIorMRCP
(magneticresonancecholangiopancreatography).Thisallowsforthe
possibilitythatamylasemightbe<3timestheULNinacutepancreatitis.
Inacutepancreatitis,serumamylaseactivitytypicallyincreaseswithin2
12hofonsetofsymptoms,hasa24hpeakandremainselevatedfor37
days.Themagnitudeofelevationisnotrelatedtoseverity.However,the
greatertherise,thehighertheprobabilityofacutepancreatitis.Serial
measurementsofserumamylasehavelittleifanyvalueinassessing
prognosisorinalteringpatientmanagement.However,ifserumamylase
remainselevatedforseveralweeks,thismayindicatepersisting
Copyright Association for Clinical Biochemistry 2012

6.2.

7.

pancreatic/peripancreaticinflammation,blockageofthepancreaticduct,
ordevelopmentofapseudocyst.

Confoundingfactors
Diagnosticspecificityislow(70%),sincePtypeamylaseisincreasedina
numberofotheracuteintraabdominaldisordersandStypeamylasein
otherconditions,e.g.salivarydisease,ovarianandlungtumours,and
rupturedectopicpregnancy.Bothmaybeincreasedindiabetic
ketoacidosis,renalfailure,HIVandmacroamylasaemia.
Thepeakofserumamylaseactivitydoesnotcorrelatewiththeseverity
ofacutepancreatitis.
Increasedamylaseactivityinserumcanresultfromaconditionknown
asmacroamylasaemiaastateinwhichamylaseformsmacromolecular
complexes,usuallywithimmunoglobulin(IgAorIgGinmostcases),but
alsoasselfpolymerisationorassociationwithotherproteins.These
complexesgenerallyretainenzymaticactivitybutcannotbefilteredby
renalglomerului;thisleadstodelayedclearanceandincreasedserum
amylaseactivity.Thisbenignconditionhasbeenreportedinasmanyas
1.5%ofhospitalisedpatients,accountingforasmuchas28%ofchronic,
otherwiseunexplainedhyperamylasaemia.Macroamylasaemiahasa
diseaseassociationwithautoimmunity,malignancy,cardiovascular
disease,diabetesmellitusandmalabsorptivedisorders.

Causesandinvestigationofabnormalresults

7.1
Highvalues
7.1.1 Causes
Pancreaticdisease:causingraisedPtypeamylasee.g.pancreatitis,
trauma,tumoursetc.
Anyotherintraabdominalcondition:e.g.biliarytractdisease,
obstruction,liverdisease,mesentericinfarction,acuteappendicitis,
tumoursetc.
Salivaryglanddisease:causingraisedStypeamylasee.g.infection,
trauma,irradiation.
Manyotherconditions:e.g.tumoursofthetestes,ovary,prostate,
oesophagus,thymus,thyroidorlung,rupturedectopicpregnancy,and
renaldiseaseincludingrenalinsufficiency
Miscellaneousconditions:includingHIV,diabeticketoacidosis,
macroamylasaemia,andvariousdrugs(e.g.opiates,diuretics,
steroids).
7.1.2 Investigation
Numerousinvestigationscanbecarriedoutincludingimagingandsecond
linebloodtestsdependingontheclinicalfeaturesofthepatient.Noother
laboratoryinvestigationscontributetothediagnosisofacutepancreatitis
(see6.1(1)butinacutepancreatitis,biochemicalabnormalitiescan
includeuraemia,hypoalbuminaemia,hypocalcaemia,hyperglycaemia,
metabolicacidosis,hypoxaemia,andabnormalliverfunctiontests.
Innonacutesituations,macroamylasaemiacanbeexcludedbyserum
electrophoresisorprecipitationmethods.

7.2 Lowvalues
7.21 Causes

Copyright Association for Clinical Biochemistry 2012

AdecreasedserumPtypeamylaseactivity(belowthelowerlimitof
normal)ishighlyspecificforexocrinepancreaticinsufficiency.
7.2.2 Investigation
Measurementofimmunoreactivetrypsinogenorfaecalelastase,aswellas
imagingofthepancreasandductscancontributetothediagnosisof
exocrinepancreaticinsufficiency.

7.3
Notes
Substantialelevationsinserumamylaseactivity(morethantwotimesthe
upperlimitofnormal)may,forreasonsthatarenotwellunderstood,be
lesscommoninpatientswithhypertriglyceridaemiaassociated
pancreatitis.

8
Performance

8.1
Sensitivity,specificityetc.forindividualconditions
Foracutepancreatitis,totalamylaseactivityabovetheupperlimitof
normalhasasensitivityof95100%,aspecificityof70%,positive
predictivevalueof1572%andnegativepredictivevalueof97100%.Ifa
cutofflevelof1000U/Lischosen,thespecificityoftotalamylase
approaches95%butthesensitivityisaslowas61%.Sensitivityisalso
reducedbylatepresentation,hypertriglyceridaemiaandchronic
alcoholism.
Thelackofspecificityoftotalamylaseactivityforacutepancreatitishas
ledtointerestinthedirectmeasurementofPtypeamylaseforthe
differentialdiagnosisofacuteabdominalpain.AnelevatedPtype
amylasehasasensitivityof84100%,aspecificityof4097%,positive
predictivevalueof5096%andnegativepredictivevalueof70100%.If
acutoffofthreetimestheupperlimitofnormalisused,thespecificityof
Ptypeamylaseis>90%.MeasurementofPtypeamylasealsoimproves
sensitivityinthelatedetectionofacutepancreatitissincePtypeamylase
remainselevatedforlongerthantotalamylaseactivity.

9.Systematicreviewsandguidelines

9.1 Systematicreviews

Noneidentified

9.2 Guidelines
BanksPA,FreemanML.PracticeParametersCommitteeoftheAmerican
CollegeofGastroenterology(2006)PracticeGuidelinesinAcute
Pancreatitis.AmJGastroenterol2006;101:23792400.
UKWorkingPartyonAcutePancreatitis.UKguidelinesforthe
managementofacutepancreatitis.Gut2005;54(III):iii1iii9.
ToouliJ,BrookeSmithM,BassiCetal.Guidelinesforthemanagementof
acutepancreatitis.JGastroenterolHepatol2002;17Suppl:S15.

9.3 Recommendations
CarrollJK,HerrickB,GipsonT,LeeSP.AcutePancreatitis:Diagnosis,
Prognosis,andTreatment.AmFamPhysician2007;75:15131520.
KiriyamaS,GabataT,TakadaTetal.Newdiagnosticcriteriaofacute
pancreatitis.JHepatobiliaryPancreatSci2010;17:2436.

Copyright Association for Clinical Biochemistry 2012

10
Links

10.1Relatedanalytes
1.Lipase
Serumlipaseisderivedmainlyfrompancreaticacinarcells.Measurement
ofserumlipaseactivitycanbeusedasanalternativetoamylaseto
supportadiagnosisofacutepancreatitis.Serumlipaseusuallyrises48h
aftertheonsetofsymptoms,peaksat24handremainselevatedfor814
days.Sincethepancreasisthemainsourceoflipase,italsohassuperior
sensitivity,specificityandgreateroverallaccuracycomparedtototal
amylase.Thesensitivityofserumlipaseforthediagnosisofacute
pancreatitisrangesfrom85100%.However,lipaseistechnicallymore
difficulttomeasureandnonspecificelevationsoflipasehavebeen
reportedinalmostasmanydiseasesasamylase.Inaddition,the
magnitudeofincreaseinserumlipasecanvarywidelydependingonthe
methodusedformeasurement.Recenttechnicalimprovementssuchas
inclusionofcolipaseincommerciallyavailablekitshasimprovedthe
diagnosticaccuracy.
Serumlipasemeasurementcanbeusefulinsuspectedacutepancreatitis
whereanotherconditionthatcausesanincreaseinStypeamylaseisalso
present.Italsopossessesahighersensitivityinalcoholinducedacute
pancreatitis.
Mostlaboratoriescontinuetomeasureserumamylaseactivityowingto
theincreasedcostsandlimitedadditionalbenefitsinthemajorityof
patientswithacutepancreatitis.

10.2Relatedtests
Urinaryamylase:hyperamylasuriaoccursinacutepancreatitis,because
thenormal3%amylase:creatinineclearanceratio(ACCR),indicatingthat
approximately3%offilteredamylaseisexcreted,increasestoabout10%.
Urinaryamylaseactivityoftenreachesveryhighlevelsinsuchcasesand
persistsforlongerthanaraisedserumamylase.However,measurement
ofamylaseinurineisnotsufficientlyaccuratetodistinguishacute
pancreatitisfromotherintraabdominalconditionsthatareassociated
withahighserumamylase.Otherthantodiagnosemacroamylasaemia,
urinaryamylaseoffersnoadvantageoverroutineserumamylase
measurement.

Author:SophieHepburn

Copyright Association for Clinical Biochemistry 2012