Development of metallothionein-based assays for the measurement of zinc status in humans PDF
Preview Development of metallothionein-based assays for the measurement of zinc status in humans
DEVELOPMENTOFMETALLOTHIONEIN-BASEDASSAYSFORASSESSMENT OFZINCSTATUSINHUMANS By VICKIK.SULLIVAN ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT OFTHEREQUIREMENTSFORTHEDEGREEOF DOCTOROFPHILOSOPHY UNIVERSITYOFFLORIDA 1997 Copyright1997 by VickiK.Sullivan Tomyparentsandmyhusband. ACKNOWLEDGMENTS IthankmymentorDr.RobertCousinsforallowingmetolearnandaccomplish exactlywhatIhadhopedtowhenIarrivedherefouryearsago,forexposingmetoa laboratorythatoffersavastarrayoftechniquesinbasicandappliedscience,andfor providinganunlimitedamountofhistime. Ialsothankmycommitteemembers,Drs.Gail Kauwell,SusanS.Percival,RachelShiremanandKuo-JangKaofortheirexcellent expertiseandguidancethroughoutthisexperience. IalsothankDr.Cousinsfor surroundingmewithagroupoftalentedandcompetentco-workers. Ithankthefollowing peoplefortheirsupport: BarbaraDavis,ChristinaKhoo,LorraineLangerhan-Foster, SteveDavis,andKarolSmithforsharingthisexperiencewithme;MelissaTuasonforher helpwiththehumanstudy;Drs.RayBlanchard,NoraHalloquistandRobertMacMahon fortheirencouragementandexpertise;WarrenClarkforputtingupwithsharingalab benchwithme,andVirginiaMauldinforalwaysgoingtheextramile. Ithankbothofmyparentsfortheirunconditionallove,andforteachingmeby examplethevalueofhardwork,persistence,andhonesty. Ialsothankthemforalways listening. IthankmyhusbandShawnforhisloveanddevotion,foragreeingtoridethis roller-coaster(graduateschool)withmeforsomanyyears,andforstayingontheride throughalltheupsanddowns. IalsothankmyfurryfriendsSpot,ChewyandPumpkin, formakingmelaughwhennooneelsecould,andforremindingmethatthesimplethings inlifearetrulythemostimportant. IV TABLEOFCONTENTS ACKNOWLEDGEMENTS iv LISTOFFIGURES vii ABBREVIATIONS » ABSTRACT x CHAPTERS 1 INTRODUCTION 1 2 REVIEWOFTHELITERATURE 5 Zinc 5 DistributionandFunctions 5 AbsorptionandTransport 6 Sources.BioavailabilityandRequirements 7 Deficiency 8 Toxicity 9 AssessmentofZincStatus 10 H Metallothionein MetallothioneinfortheMeasurementofZincStatus 13 ReverseTranscriptasePolymeraseChainReaction 13 3 MATERIALSANDMETHODS 17 RefinementofHumanMetallothioneinELISA 17 CompetitiveELISA 17 ELISAPlates 17 SourceofMetallothionein 17 PrimaryAntibody 19 ErythrocyteLysatePreparation 23 ProteinQuantitation 24 PlasmaZincandCopper 24 SandwichELISA 24 DevelopmentofCompetitiveReverseTranscriptasePolymeraseChain ReactionAssay 26 CellCulture 25 PreliminaryHumanStudy 26 IsolationofMonocytes 26 ExtractionandPurificationofRNA 27 ReverseTranscriptasePolymeraseChainReactionAssays 28 CompetitiveRT-PCR 29 LongitudinalHumanStudy 35 ExperimentalDesign 36 AnalyticalProcedures 36 StatisticalAnalysis 37 4 RESULTS RefinementofHumanMetallothioneinELISA 41 ELISAPlates 41 Antibodies 41 ErythrocyteLysatePreparation 42 SourceofMetallothionein 42 PreliminaryExperimentsforCompetitiveRT-PCRAssayandSandwich ELISA 50 THP-1Cells 50 FlowCytometry 50 CompetitiveRT-PCR 50 SandwichELISA 54 LongitudinalHumanStudy 54 CompetitiveRT-PCR 54 SandwichELISA 55 PlasmaZinc 55 PlasmaCopper 56 5 DISCUSSIONANDCONCLUSIONS 62 APPENDIXA 73 APPENDIXB 80 REFERENCE 81 BIOGRAPHICALSKETCH 92 VI LISTOFFIGURES Figure gage. 3-1. Diagramofcompetitivereversetranscriptasepolymerasechainreaction(RT- PCR)assay 31 3-2. Overviewofmetallothionein(MT)cDNAcompetitortemplateconstruction 32 3-3. Themetallothionein(MT)cDNAandcompetitorcDNAwereresolvedonan 8%polyacrylamidegelstainedwithethidiumbromide 33 3-4. Competitivereversetranscriptasepolymerasechainreaction(RT-PCR)assay toquanifymetallothionein(MT)mRNA 34 3-5. Overviewofprotocolforlongitudinalhumanstudy 38 3-6. Competitivereversetranscriptasechainreaction(RT-PCR)assaytoquantify metallothionein(MT)mRNA 39 3-7. PercentintegratedintensityforcDNAbandsaftercompetitiveRT-PCR 40 4-1. TitrationCurveforAnti-HumanMetallothioneinChickenAntibodies 43 4-2. SandwichELISAtoTestforHumanMetallothioneinSpecificity 44 4-3. SandwichELISAforComparisonofMonoclonalAntibodies 45 4-4. DirectELISAfortheComparisonofMTFromDifferentSpecies 46 4-5. EffectofProteaseInhibitorsonErythrocyteLysateMTStabilityOverTime 47 4-6. Zinc-stimulatedincreaseinmetallothioneinmRNAlevelsinTHP-1cellsusing semi-quantitativereversetranscriptasepolymerasereaction(RT-PCR) 48 4-7. Flowcytometryprofileofhumanmonocytes 49 4-8. HumanMonocyteMTcDNAafterCompetitiveRT-PCR 51 4-9. SandwichELISAforErythrocyteMetallothionein 52 vn 4-10. StandardCurveforSandwichELISA 53 4-11. CompetitiveRT-PCR: MonocyteMetallothioneincDNA 57 4-12. Polyacrylamidegelelectrophoresisshowingintra-assayvariationfor competitiveRT-PCRassay 58 4-13. Erythrocytehumanmetallothionein(hMT)asanalyzedbysandwichELISA 59 4-14. PlasmaZinc 60 4-15. CopperZinc 61 5-1. ComparisonofMonocyteMTmRNAandErythrocyteMTproteinResponse toZincSupplementation 70 Vlll ABBREVIATIONS bp basepair BSA bovineserumalbumin cDNA complementarydeoxyribonucleicacid ddH2 glassdistilleddeionizedwater DEPC diethylpyrocarbonate DMEM Dulbecco'smodificationofEagle'smedium DNA deoxyribonucleicacid dNTP deoxynucleosidetriphosphate ELISA enzyme-linkedimmunosorbentassay FAA flameatomicabsorption FACS fluorescenceactivatedcellsorter FBS fetalbovineserum FITC fluorosceinisothyocyanate FPLC fastproteinliquidchromatography IACUC InstitutionalAnimalCareandUseCommittee ICBR InterdisciplinaryCenterforBiotechnologyResearch IgG immunoglobulinG hMT humanmetallothionein mRNA messengerribonucleicacid MT metallothionein NIH NationalInstituteofHealth PAGE polyacrylamidegelelectrophoresis PBS phosphate-bufferedsaline PCR polymerasechainreaction RIAs radioimmunoassays RT-PCR reversetranscriptasepolymerasechainreaction IX AbstractofDissertationPresentedtotheGraduateSchool oftheUniversityofFloridainPartialFulfillmentofthe RequirementsfortheDegreeofDoctorofPhilosophy DEVELOPMENTOFMETALLOTfflONEINBASEDASSAYSFORTHE MEASUREMENTOFZINCSTATUSINHUMANS By VickiK.Sullivan May,1997 Chairman: RobertJ.Cousins MajorDepartment: FoodScienceandHumanNutrition Whilezincisanessentialmineralwithnumerousphysiologicalroles,areliable indexforzincstatusinhumanshasnotbeendeveloped. Metallothionein(MT),acysteine- richproteinoflowmolecularweight,isinducedbyzinc. BecausetheMTgeneis transcriptionallyregulatedbyzinc,MThasgreatpotentialforservingasanindexof dietaryzincstatus. Thespecificaimsofthisprojectwereasfollows: 1)todevelopanew enzyme-linkedimmunoassay(ELISA)forthemeasurementofhumanMT(hMT)in erythrocytes;2)todevelopamethodformeasuringhMTmRNAlevelsinmonocytes;and 3)totestthesemethodsinahumanzincsupplementationstudy. AsandwichELISAwas developedforthemeasurementofhMTinerythrocytes. Acompetitivereverse transcriptasepolymerasechainreaction(RT-PCR)assaywasdevelopedforthe measurementofmonocytehMTmRNA. Twenty-fivehealthymalesubjectsparticipatedin an18dayzincsupplementationstudy. ErythrocytehMT,monocytehMTmRNA,plasma zincandplasmacopperwereevaluated. Zincsupplementationresultedinasignificant
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