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1、Experiment10DeterminationofuricacidinhumanserumObjective:After1earningthiscourse,studentsshou1dgrasptheprincip1eforthequantitativedeterminationofuricacidinb1oodserumanditssignificanceforthec1inica11ydiagnosticuse.Princip1eUricacidisthewasteproductproducedfromthedegradationofpurines.Itisproducedbythe
2、actionofxanthineoxidaseonxanthineandhypoxanthinewhichareproductsofnuc1eicaciddegradation.Whenuricacidispresentinabnorma11yhighconcentrationsintheb1ood,ittendstocrysta11izeoutinthebodyjoints,causingaverypainfu1inf1ammatoryconditionknownasgout.Increased1eve1sofuricacidarea1soassociatedwithrena1fai1ure
3、and1argedietaryintakeofpurines.Uricaciddeterminationisthusimportantandusefu1forc1inica1diagnosis.Thec1assicchemica1methodforthedeterminationofuricacidisbaseduponthereductionofphosphotungsticacidbyuricacidtoab1uephosphotungstatecomp1ex.Themethodisnotspecific.In1980Fossatidescribedaprocedureforassayin
4、guricacidusinguricase.Theprocedureismorespecific.ThehydrogenperoxideformedbytheactionofuricaseonuricacidreactswithyV-ethy1-/V-(2-hydroxy-3-su1fopropy1)-3-methy1ani1ine(TOOS)and4-aminoantipyrineinthepresenceofperoxidasetoformaredco1oredquinoneiminedye.Uricacidisvirtua11yinso1ub1einwaterorcommonorgani
5、cso1vents,butitcanbedisso1vedinbasicso1utionsuchasaqueousso1utionsofNa2CO3,NaOHandammoniumhydroxide.UricaseUricacid+H2O+O2A11antoin+CO2+H2O2Peroxidase2H2O2+4-aminoantipyrine+TOOSQuinoneimine+4H2OTheintensityoftheco1oredproductisdirect1yproportiona1totheconcentrationofuricacidinthesamp1e.Theconcentra
6、tionofuricacidisdeterminatebytheco1orimeterwithmaximumabsorbanceat550nm.Theuricacidassayisca1ibratedbyreferencingtheabsorbanceoftheunknownsamp1etotheabsorbanceoftheca1ibrator.EXPECTEDVA1UESMa1es:208-428:mo1/1(3.5-7.2mgd1)Fema1es:155-357:mo1/1(2.6-6.0mgd1)ApparatusandReagents1. Spcctrophotomctcrorco1
7、orimeter.Anyinstrumentcapab1eofreadingabsorbanceaccurate1ywithasensitivityof0.001absorbanceat550nmmaybeused.0.5cmcuvettesoraf1owce11capab1eoftransmitting1ightat550nm.2. Reagentsnecessaryforthedeterminationofuricacidareinc1udedinthekit(10X5m1)Theworkingreagent,uricacidreagent(R1)contains,afterreconst
8、itutionwiththebuffer(R2):peroxidase1800U/1Iuricase600U/1ascorbateoxidase1000U/1R，TOOS1.4mM4-aminoantipyrine0.3mMJBuffer(pH7.8),preservatives,andstabi1izersR2Procedures1. Preparetherequiredvo1umeofworkingreagent.Theworkingreagentispreparedbyadding5m1ofbuffer(R2)toabott1eofuricacidreagent(RI),mixatonc
9、e.2. Prepare3dryc1ean1ubes,anddoasfo11ows:numberreagents(m1)b1ankStandardsamp1esamp1e(1)(2)(3)Standarduricacid1iquid(5mg/d1)00.020Serumsamp1e000.02Disti11edwater0.0200Workingreagent1.01.01.0Mixatonce!3. Incubatefor10minutesat37oCanddeterminetheabsorbanceoftheca1ibrator(As)andofserum(A)at550nmusingth
10、edisti11edwa1ersamp1easthereagentb1ank.4. Theca1cu1atetheuricacidconcentrationofthesamp1eA550ofthetubefortestingScrumuricacidconcentration(mg/d1)=concentrationofstandardA55oofthetubeforstandarduricacid(mg/d1)Question1. Whatstheprincip1eforthequantitativedeterminationofuricacidinb1oodserum?2. Whatsthec1inica1significanceofuricaciddetermination?
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