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bioassay of acth 5ht histamine

bioassay of acth 5ht histamine

Tajuddin
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bioassay of acth 5ht histamine

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  1. BIOASSAYOFd-TUBOCURARINE, HISTAMINE, 5-HT & ACTHBy Tajuddin Shaik

  2. BIOASSAYOFd-TUBOCURARINE 1.RabbitHead-dropMethod: Principle: d-TubocararineHClisinjectedintothemarginalveinofarabbit’seartillthe rabbit’sneckmusclesarerelaxedsuchthattheanimalcannotholditshead up.Thetotalamountoftestsamplerequiredtoproducetheendpointis comparedwiththetotalamountoftheSTDsamplerequiredtoproduce similarendpoint. Selectionof Rabbits: Rabbitsweighing2kgareused.Animalsshouldbefreefromdisease, obtainedfromahealthycolonyandshouldbeaccustomedwiththe experimentalprocedure.

  3. BIOASSAYOFd-TUBOCURARINE • ExperimentalProcedure: • Rabbitisplacedinaholderwithitsheadprotrudingoutside.Thehead shouldbefreelymovable. • Minimum8rabbitsareused.Theyaredividedinto2groupseach containing4rabbits. • FirstgroupwillreceiveSTDsampleandthesecondgroupwillreceivethe sampleundertestd-Tubocurarinesolutionisinjectedataconstantspeed byinfusionapparatusthroughthemarginalvein. • Injectionshouldbegivenatarateof0.4ml/minandshouldtakeabout10 min.Dose0.012%w/vinsaline.

  4. BIOASSAYOFd-TUBOCURARINE • Infusioniscontinuedtilltherabbitwillnotbeinapositiontoholdits headerectortherewillbenoresponsebyfocusinglightontheeyes. Rabbitsrecoverimmediatelyfromtheeffectofcurarization. • Duringtheexperimentthereisapossibilityofrespiratoryembarrassment whichistreatedbyinjectingNeostigminemethylsulphate(0.05mg.)and atropinesulphateimmediatelythroughthemarginalearvein. • Cross-overtestiscarriedouttominimizebiologicalerrorduetoanimal variation. • ThoserabbitswhichreceivedtheSTDsampleonthefirstdaywillbe giventestsampleontheseconddayofexperiment andviceversa. • Meandosewhichproducesheaddropofthetestsampleiscompared withthemeandoseofstandardpreparation.

  5. BIOASSAYOFd-TUBOCURARINE • FrogsRectusAbdominusmusclePreparation: • Afrog ispithedandlaidon itsbackonacorkcovered board to whichitis pinned.Theskincoveringtheabdomeniscutawayandtherectus abdominusmuscleofonesideisdissected. • Themuscleisthenpinnedtothecorkbyfourpinstokeepitsnormal lengthwhileathreadissewnthrougheachend. • Itisthenmountedintheorganbathcontainingfrog'sRingersolution whichcontains:NaCl,6.5gm.;KCl,0.29gm.;CaCl2,0.24gm.;NaHCO3, • 0.4gm.;glucose,1.5gm.anddistilledwater2000ml. • Oxygenationiscarriedouttokeepthetissuealive.Themuscleis stabilizedfor30-45min.inordertogetcriticalquantitativeresponse.

  6. BIOASSAYOFd-TUBOCURARINE • FrogsRectusAbdominismusclePreparation: • Theresponsesarerecordedusingisotonicfrontalwritingleverwith1g tension. • TwosimilarcontractionswiththesameconcentrationofAchare obtained. • ThreedosesoftheSTDsampleandoneintermediatedoseofthetest sampleareselected&thereductioninheightofcontractioninducedby Achisnoteddown. • Achcontractionisrecordedonslowmovingdrumfor90sec.d- Tubocurarineisallowedtoactfor30sec. • Thepercentagereductionateachdoselevelsiscalculatedandlogdose responsecurveoftheSTDdrugisplotted.Alinearresponsewillbe obtained.Thepotencyoftestsampleiscalculatedfromthestandard curve.

  7. BIOASSAY OFHISTAMINE

  8. BIOASSAYOFHISTAMINE • Histamineispresentinalmostalltheanimaltissuesmostlywithinmastcell& basophilgranules. • Tissuesrichinhistamineareskin,gastricandintestinalmucosa,lungs,liver& placenta. • Non-mastcellhistamineispresentinbrain,epidermis,gastricmucosa& growingregions. • Histaminealsoexertsavariousotherimmuneregulatoryfunctionsby modulatingthefunctionsofmonocytes,Tcells,macrophages,neutrophils, eosinophils,Bcells&dendriticcells. • Histamineisalsopresentinblood,mostbodysecretions,venoms&pathological fluids.Itisnowknowntoplayimportantphysiologicalroles.Histaminereceptors (H1,H2,H3&H4)presentontargetcells. • Bioassayofhistamineonisolatedguineapigileumcanbedetermined bygraded bioassayprocedurei.e.i)Matchingbioassay,ii)Interpolationbioassay,iii) Bracketingassay,iv)Multiplepointassays.

  9. Bioassayofhistamineinbiologicalsamplescanbestudiedbyusingdifferent bioassaymethods.Dependinguponpharmacologicalaction,histaminecanbe assayedby: • Contractileeffectofisolatedileumofguineapig, • Contractileeffectofuterusofguineapig,and • Fallinbloodpressureofanaesthetizedandatropinizedcat • Bioassayofhistamineusingguineapigileum: • Principle • Guineapigileumisveryusefulpreparationforthebioassaymethods.Itismore • sensitivetohistamine.Contractileresponseofhistaminetoileumisdue to presenceofH1receptor.

  10. PreparationofStandardandothersolution: • PreparethestockofTyrodesolution.Alsopreparethestandardstock solutionofhistamine(1mg/ml)&thendifferentconcentrationsof histaminebyserialdilutionmethod. • Procedure: • Sacrificethe24hrfastedguineapigbystunningonthehead. • Fixtheanimalonthedissectingboardbytyingitslegs. • Opentheabdominalcavitybyasmallhorizontalcutfollowedbyvertical midlineincisionandexposetheabdominalorgans.

  11. Trace theileocaecal junctionbyliftingthe caecum,thengoupwardsupto8cm fromthejunctionandcutthe2-3cmlongsegmentofileummuscle(excluding theterminal5-8cm,containsexcessofexcitatoryα-adrenergicreceptor,which mayinterfereinthestudy). • ImmediatelyplaceitinapetridishcontainingaeratedwarmTyrodesolution. Slowlyremovethemesenteryattachedtothemuscle&thengentlycleanthe lumenofileumbypassinglukewarmTyrodesolutionthroughitwithapipette orsyringe. • MountthepreparationintheinnerorganbathcontainingTyrodesolution(20 ml)maintainedat37ºC.Tiethebottomendofthemuscletothehookof aerationtubeandtheupperendtotheisotonicfrontalleverbyathread withoutclosingthelumen.

  12. Adjustthemagnificationofresponseto7-10fold.AeratethetissuewithO2 orcarbogenslowly(40-60bubblesperminutes). • Stabilizethetissuefor30minbyapplyingatensionof0.5gweightattached tothelever,duringwhichwashthetissuewithfreshTyrodesolutiononcein every10min. • Use5mintimecyclewithcontacttimeof30secforrecording the contraction dependentresponsesoftissueduetohistamineonthesmokeddrums. • RecordtheresponsesofSTD&testcompounds. • Recordtheresponsesoftestcompoundi.e.unknownconcentrationof histaminewithgraduallyincreasingvolume,tillobtainingaresponse(T)which wouldlieonthelinearportion(30-70%)oftheCRC. Fix theresponseobtained duetovolumeofT.

  13. RecordthegradedresponsesofSTDsolution&testsolutionofhistamine.RecordthegradedresponsesofSTDsolution&testsolutionofhistamine. • Thepotencyofthetestsampleiscalculatedinrelationtothatofthestd. preparationbydividingtheaveragelethaldoseofthesampletothetestand expressedasunitspergram. • Potency =LethaldoseofSTD/Lethaldoseoftest=unit/g.

  14. BIOASSAYOF5-HT

  15. BIOASSAYOF5-HT • 5-Hydroxytryptamine(5HT)isthebiologicallyactivelocalhormone,low molecularweight&alsoanimportantneurotransmitterinthebrain& periphery. • Itwasfirstdetectedinserum(serotonin)&enterochromaffincellsofgut mucosa(enteramine)&laterbothwereidentifiedtobe5HT.Todaytheterms 5HT&serotoninareusedinterchangeably. • About90%ofbody'scontentof5-HTislocalizedintheintestines;mostofthe restisinplatelets&brain. • Itisalsofoundinwaspandscorpionsting,andiswidelydistributedin invertebratesandplants(banana,pear,pineapple,tomatoetc.). • Theconcentrationof5-HTinbiologicalsamplescanbeassayedby: 1. 2. 3. Isolatedfundusstripofratstomach, Isolatedterminalcolonofrat, Isolatedratuterus&4. Perfusedrabbitear.

  16. Isolatedfundusstripofratstomach Principle: • Ratfunduspreparationisaverysensitivetissueforthebioassaymethods& isusefultostudytheactionofseveralsubstanceslike5-HT,ACh,PGE2& bradykinin. • Fundusstrippreparationisslowcontractingmuscle. • Funduspartofstomachcanbeeasilyidentifiedbyitsgreycolour&situated abovethepinkcolouredthickpyloricpart. • Azig-zagpreparationofthefundalstripispreparedsoastoexpose maximumportionofthetissuetodrug. • PreparationofStandardandothersolution: • PreparethestockofKrebssolution.PreparetheSTDstocksolutionof serotonin(1mg/ml)&thendifferentconcentrationsofserotoninbyserial dilutionmethod.

  17. BritishJournalofPharmacology,Volume:120,Issue:S1,Pages:142-147,First published:08September2011,DOI:(10.1111/j.1476-5381.1997.tb06791.x)

  18. Procedure: • Sacrificethe24hrfastedratbystunningonthehead.Fixtheanimalonthe dissectingboardbytyingitslegs.Opentheabdominalcavitybyasmall horizontalcutfollowedbyverticalmidline incisionandexposetheabdominal organs. • Identifythestomachandseparateitfromtheabdomenbygentlycuttingits cardiacandpyloricend.PlaceitinapetridishcontainingaeratedwarmKrebs solution. • Incisethefundusofthestomach(uppergreypart)frompyloricpart(pink andthickpart).Cutthefundusfromthelessercurvatureandopenit longitudinally.Thencutthefundusatthemidlineintotwoequalparts.Give alternatetransversecuts(zig-zagcut)onoppositesidesofthemuscleto makeafundalstrippreparation.

  19. MountthepreparationintheinnerorganbathcontainingKrebssolution(20 ml)maintainedat37ºC.Tiethebottomendofthemuscletothehookof aerationtube&theupperendtotheisotonicfrontalleverbythread.Adjust themagnificationofresponseto10-15fold.AeratethetissuewithO2or carbogenslowly(40-60bubbles perminutes). • Apply1gload&stabilizethetissuefor30minduringwhichwashthetissue withfreshKrebssolutiononceinevery10min. • Use a5mintimecyclewith contact timeof90s(sincethe fundusstrip muscle contractsslowlyandrelaxesslowly)forrecordingthecontractiondueto serotonin. • RecordthegradedresponsesofSTDsolution& testsolutionofserotonin. • Thepotencyofthetestsampleiscalculatedinrelationtothatofthestd. preparationbydividingtheaveragelethaldoseofthesampletothetestand expressedasunitspergram.

  20. BIOASSAYOFACTH

  21. ACTH(Adrenocorticotropichormone,Corticotropin)ispolypeptidetropic hormone(39aminoacids)secretedbytheAnteriorPituitaryGland. • ACTHstimulatestheproductionofCortisolasteroidhormoneimportant forregulatingGlucose,Protein&LipidMetabolism,suppressingtheimmune systemresponse&helpingtomaintainBloodPressure. • OfficialPreparations: • Corticotropininjection:Isasterilesolution,inasuitablediluent,ofthe polypeptidefromthepituitaryglandsofmammals.Potency rangeshouldbe • 80.0–120.0%ofUSPcortiotropinunits. • Corticotropinforinjection,antimicrobialagent.Repositorycorticotropin injectioniscorticotropininasterilesolutionofpartiallyhydrolyzedgelatin&is intendedforS.C.&I.Mrouteuse.Thissolutionhasbeenadoptedasthe referenceSTDforthebioassay.

  22. Packing:Preserveinsingle-doseormultiple-dosecontainersofType-1glass.Packing:Preserveinsingle-doseormultiple-dosecontainersofType-1glass. Storage:Storeincoldplace. Labeling:Injectionrecommendsintravenousadministration. Purposeandrationale: Thisisahistoricalassaymethod. AdministrationofpituitaryACTHdecrease theascorbicacidpresentintheadrenals.Thedepletionofadrenalascorbic acidisafunctionofthedoseofACTHadministered.Thisrelationship hasbeenusedforaquantitativeassayofACTH.

  23. Solutions: SolutionA:Fiveunitsoftestorstandarddissolvedin0.25mlof0.5%phenol solutionanddilutedwith8.1mlof15%gelatinsolution(Now0.5mlcontain 300mUACTH). SolutionB:Threemlof solutionAdilutedwith6ml gelatinsolution.Nowconcentrationreducedto100mUACTH/0.5ml. SolutionC:Again3mlofsolutionBdilutedwith6mlofgelatinsolution,the resultingsolutioncontains33mUACTH/0.5 ml.

  24. Procedure • MaleWistarrat(100-200g)arehypophysectomized(pituitary gland • removedbysurgery)onedaypriortothetest. • Foronetestwith3doseofTestpreparationandSTDsolutionusedforthe study. • Numberofhypophysectomizedratsrequired:atleast36(preferably 60). • Thehypophysectomizedratsarerandomlydistributedintosixgroups.Each ratreceivessubcutaneous0.5mlofthevariousconcentrationsoftestor standard.

  25. Procedure • Threehoursafterinjection,theanimalsareanesthetizedandbothadrenals removed, freedfromextraneoustissueandweighed. • Theratsaresacrificedandthescullopenedtoverifycompletenessof hypophysectomy. • Theadrenalsarehomogenizedinglasstubescontains200mgpuresand& • 8.0mlof4%trichloroaceticacidandtheascorbicaciddetermined(Roeand Kuether,1943). • Thepotencyratioincludingconfidencelimitsiscalculatedwiththe3+3 pointassay.

  26. Ascorbicaciddetermination: • Reagents • 0.02%ascorbicacidsolution • 85%sulfuricacid(9N H2SO4) • 0.02g/mlofdinitrophenolhydrazinein9NH2SO4 • 0.06g/mlofthioureaaredissolvedindistilledwater • Charcoal • Preparationof0.02% ascorbicacidsolution • 100mgL-ascorbicacidaredissolvedin100mlof4%trichloroaceticacid (1mg/mlsolution)(SolutionA= 1%solution) • 2mlofSolutionAdilutedin10mlof4%trichloroaceticacidtoachievea 0.2%ascorbicacidsolution(solutionB) • 1mlofsolutionBdilutedin10mlof4%trichloroaceticacidtoachievea 0.02%ascorbicacidsolution(solutionC)

  27. Preparationofothersolutions • Sulfuricacid(85%)isobtainedbyadding900mlconcentratedsulfuric acidto100mldistilledwater. • Twogmdinitrophenolhydrazinearedissolvedin100ml9NH2SO4(75ml distilledwater&25mlconcentratedsulfuricacid). • Sixgmthioureaaredissolvedin100mldistilledwater. • Calibration • Trichloroaceticacid(4%)isaddedto0.0,0.5,1.0,2.0,3.0,4.0,6.0,8.0ml ofthe 0.02%ascorbicacidsolution(solutionC)and1.0, 1,5and2.0 mlof the0.2%ascorbicacidsolutiontoreachafinalvolumeof8.0ml(Solution B). • 100mgcharcoalisaddedtoeachsampleandthoroughlymixedby shakingfor1min. • After5minthesolutionsarefiltered

  28. Ascorbicaciddetermination: • Analiquotof0.1mlofthe6%thioureasolutionisaddedto2.0mlofthe filtratefollowedby0.5mldinitrophenylhydrazinesolution. • Themixtureisshakenandheatedfor45minat57°Cinawaterbath. • Thesolutionsareplacedinanice-coldwaterbathandwithfurthercooling • 2.5mlofthe85%sulfuricacidareadded. • Thecalibrationcurveisestablishedatawavelengthof540nmusingthe solutionswithoutascorbicacidasblank.

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