血清酮体总量、D-3羟丁酸-乙酰乙酸-测定方法学研究-宋晓冬

:1007-4287(2005)03-0415-03(D-3+)宋晓冬1,孔凡军1,魏宇鹏1,丁　旭1,刘淑贤1,崔建丽1,朴桂花1,朱海燕1,张　季1,刘志杰2(1.,130011;2.):　(D-3+),。　,D-3,,。　,,,D-3D-3。　D-3,,,。:;D-3;;;;:R446.1:AThestudyofdterminationmethodforthetotalacetonebodiesofserum(D-3HydroxyButyrate+CetoaceticAcid)SONGXi-ao-dong,KONGFan-jun,WEIYu-peng,etal.(TheFourthHospital,JilinUniversity,Changchun130011,China)Abstract:Objective　Inordertomonitorthediabeticketoacidosis,wemanagedtoestablishamethodwhichcanusedonthefullauto-biochemicalanalysisinstrumenttodeterminethetotalacetonebodiesofserum.Methods　Accordingtothemetabolizeprocessandthereactiontheoryoftheacetonebodiesintheserum,wemanagedtousetheenzymaticdynamicsmethodtoestablishaquantita-tivemeasurationtodiscusstherelativebetweenthetotalacetonebodiesofserumanddiabetesmellitusketoacidosis.Results　Themethodiseasytooperate.Itcanbeapplicableforthefullauto-biochemicalanalysisinstrument.Thelaboratoryindexesarealluptoscratch.ItsresulthasthehighrelativitywiththeD-3-hydroxybutyratekitofRandoxLaboratoriesLtd.ofUnitedKingdom.Conclu-sion　ComparedwiththedetermineofD-3-hydroxybutyrate,thedetermineofthetotalacetonebodiescanincreasethepositiveexam-inationrateofketoacidosis.IthasimportantsignificationsforthedoctorstofindtheDMketoacidosisandprotectthepatientfromtheDMketoacidosisasearlyaspossible.Keywords:totalacetonebodiesofserum;D-3-hydroxybutyrate;cetoaceticacid;diabetesmellitus(DM);ketoacidosis;speedanalyticalmethod(ChinJLabDiagn,2005,9:415)　　D-3,98%,。,,,。,(D-3)。:,,,,,D-3D-3:CH3-CH2-C-COOH+NADH+H+OD-3pH7.0pH8.5CH3-CH2-CH-COOH+NAD+CH(D-3)D-3,D-3D-3,NADH340nm,D-3,。1　1.1　　①Ⅰ:1.125ml+—415—　20056　9　31.125mlNADH+100u,、。②Ⅱ:Tris-HCl2.1ml+NAD3.1ml+2.8ml。1.2　　7170A。1.3　　4μl,Ⅰ:20μl,Ⅱ:200μl,,37℃,60,180,340nm,700nm。2　2.1　　,,NADH,1min,1～3minNADH。4(■AF)4(■AE),■AF■AE=0.98,0.9≤■AF■AE≤1.1。2.2　　D-3,15mmolL、14mmolL、13mmolL……1.0mmolL,20,:Y=0.9832X+0.0767,r=0.9988。0.01～10.00mmolL。1　2.3　pH　Tris100mmolL,pH7.5～9.50.2pH,2,pH8.5,pH8.3～8.7。2.4　　,4mmolLD-34mmolL,2.05mmolL。102.5%。8mmolLD-38mmolL,4.04,101%。2.5　　1mmolL100,0.01mmolL,0.01mmolL。2.6　　、、,20,:X1=0.046　CV1=1.03%X2=0.257　CV2=1.12%X3=3.526　CV3=1.25%。2.7　　D-3,、、20,,:X1=0.0398　Y1=0.0564X2=0.2595　Y2=0.3016X3=1.5240　Y3=1.7200:Y=1.0989X+0.0247,r=0.9896,X=0.60778,Y=0.69265。2.8　　、,2～8℃,。2.9　　459217,232,,25～71,,:x±s=(0.1429±0.0549)mmolL,x±s=(0.1872±0.0724)mmolL,P0.01,0.033～0.33mmolL。3　D-398%,,D-3,3-3-。pH8.5,40%D-3,NADH,340nm。,,,。,,。150UL,,;,,。D-3,,,,。:0.01mmolL0.033～0.33mmolL,102.5%101%,,。0.01～10mmolL,—416—ChinJLabDiagn,June,2005,Vol9,No.3X1=0.046,CV1=1.03%,X2=0.257,CV2=1.12%,X3=3.526,CV3=1.25%。、,。:,459,217,x±s=0.1429±0.0549mmolL,232,x±s=0.1872±0.0724mmolL,P0.01,0.033～0.33mmolL。:[1],,　,.β-[J].,2002,12(11):35.[2]　,　,,.D-[J].,2003,16(4):333.[3]EP05-A.Evaluationofprecisionperformanceofclinicalchemistryde-vice;ApprovedGuideline.NNCLS(http:)[4],,.18β-[J].,2000,20(4):243.(:2004-11-12)　:1007-4287(2005)03-0417-03RT-PCRRNA宗成国1,王晓波2,徐廷国1,于　静1,尹家俊1＊(1.,116001;2.):　RT-PCRRNA。　RNARNA,RT-PCR。　PCR,、PCR,。　RNA,。:RT-PCR;;:R446.6:ATheoptimalconditiontodetectquantificationofRNAusingsemi-quantitativereversetranscription(RT)-PCRZONGCheng-guo,WANGXiao-bo,XUTing-guo,etal.(TheaffiliatedZhongshanHospitalofDalianUniversity,Dalian116001,China)Abstract:Objective　TostudysomefactorstodetectquantificationofRNAusingsemi-quantitativereversetranscription(RT)-PCR.Methods　Underallkindofconditions,thesemi-quantativeRT-PCRtodetecttelomereasegenemRNAwasstudied.Results　InPCR,theprimerforinternalreferencegene,contentofdNTP,andruningcyclesaffecttheproductsmuchthantheannealingtemperature.Conclusion　So,inthismethod,theexperimentparametershouldbeoptimizedfirstly.Keywords:RT-PCR;reversetranscription;phosphatedehydrogenase(ChinJLabDiagn,2005,9:417)＊　　RT-PCRmRNA。RT-PCR、、[1～4],,。hTR,———(glyceraldehydephosphatedehydrogenase,GAPDH),mRNAPCR。,PCR。1　1.1　　(HeLa);(DEPC)、BICTrizolReagent、TaKaRaRNALAPCRTMKit(AMV)Ver.1.1、TaKaRaLAPCRTMKitVer.2.1;:RNA(hTR)(GAPDH)。hTR-F:5'-TA-ACCCTAACTGAGAAGGGCGTAG-3';hTR-R:5'-CG-GCTGACAGAGCCCAACTCTTCG-3';268bp。GAPDH-F:5'-TCCTCTGACTTCAA-CAGCGACACC-3';GAPDH-R:5'-TCTCTCTTCCTCTT-—417—　20056　9　3