Bioassay vasopressin digitalis ACTH

Bioassay

1. BIOASSAY OF VASOPRESSIN

2. BIOASSAY OF VASOPRESSIN
Thevasopressoractivityisestimatedbycomparingtheactivityofpreparationbeing
examinedwiththatoftheStandardPreparationofargininevasopressin(AVP)underthe
conditionsofasuitablemethodofassay.
STD
SyntheticAVPpeptideacetatewithhumanalbuminandcitricacid(suppliedinampoules
containing8.20Units),
METHOD AIP 1966
Injectslowlyintothetailveinofamalealbinoratweighingabout300gasolutionofa
suitablealpha-blocker.Forexample10ml/kgofbodyweightofasolutionpreparedby
dissolving5mgofPhenoxybenzamineHClin0.1mlofethanol(95%)+0.05mlof1M
HCl+dilutingto5ml0.9%NaCl.

3. After18hrs,anaesthetisetheratwithananaestheticthatwillmaintainaprolonged&
uniformBloodPressure.After45to60mins,tietheratonitsbacktotheoperatingtable
byitshindlegs.
Tracheaiscannulatedtomaintainrespiration&Carotidarteryiscannulatedtorecord
theBP.
Thencannulatethefemoralveininj.ofSTD&Testvasopressin
Firmlyfixawetabsorbentcottonswabtothethighsoastocovertheincision&
cannula.

4. Atthisstageinjectthroughthevenouscannula200Unitsofheparintopreventclotting
Responsesto2dosesofSTD&2dosesofTestpreparationofvasopressinaretakenwith
helpofBPapparatus.Usually3-5mUofvasopressinmaygiveappropriateriseinBP.
Basedontheresponsesobtained(4pointedgraphicalmethodbioassay)conc.of
vasopressincanbeobtained.

5. METHOD B
BasedontheAntidiureticactivityofvasopressin
SixteenMalealbinorats2groupseachgroupcontain8rats.STDgp0.006unit&
TestgpEquivalentdose
Afterinjectionanimalsareallowedtometaboliccagecollecttheurinebyusing
graduatedmeasuringcylinder.
Afterfirstcollectedurineofeachanimalvolumeisrecordedatintervalsof15minfor
3-4hrs

6. After4hrsurineflowstops&observationsarediscontinued.Time(inmin)for
excretionofthehalfthevolumeofurineiscalculatedforeachgroup.
Crossovertestiscarriedoutafter24hrs
Meanvaluesobtainedfromtheresultsof2gps.
Meanvaluesshouldbebetween96-135min.
If,bothmeantimevaluesaresamebothshouldcontainthesameunits
IfthetimesaredifferentthenrelativepotencycanbecalculatedbycomparingtheSTD.

7. BIOASSAY OF DIGITALIS

8. BIOASSAY OF DIGITALIS
PRINCIPLEPotencyofthetestsampleiscomparedwiththatofthestandard
preparationbydeterminingtheactiononthecardiacmuscle.
STDThestandardpreparationisamixtureofdried&powdereddigitalisleaves
(1unit=76mg.)
Preparation of Extracts
Exactamountofthepowderisextractedwithdehydratedalcoholinacontinuous
extractionapparatusfor6hrs.Thefinalextractshouldcontain10ml(5ml.alcohol+5ml.
water)per10g.ofdigitalispowder.Itshouldbestoredinbetween5
o
C&–5
o
C.

9. Guinea-pig Method (Endpoint method)
STD&Testsampleextractsaredilutedwithnormalsalineinsuchawaythat1gofdigitalis
powderisdilutedto80ml.
Aguineapigisanaesthetizedwithasuitableanaesthetic.Itisdissectedontheoperation
table.Thejugularveinistracedoutbyremovingadheringtissues&cannulatedbymeans
ofvenouscannula.
Apinisinsertedintheheartsuchthatitgetsinsertedintheapexoftheheart.Inthis
way,wecanobservetheheartbeatsbyupanddownmovementsofthepin.

10. Theinjectioniscontinuedthroughvenouscannulauntilltheheartisarrestedinsystole.
Theamountofextractrequiredtoproducethiseffectistakenasthelethaldoseofthe
extract.
Anothersetof19animalsofthesamespeciesareusedforthisexperiment&the
averagelethaldoseisdetermined.Lethaldoseshouldbeoccasionallychecked.
Thelethaldoseofthetestsampleisdeterminedinasimilarwayusingminimum6
guinea-pigsofthesamestrain.Thepotencyofthetestsampleiscalculatedinrelationto
thatofthestd.preparationbydividingtheaveragelethaldoseofthesampletothetest
&expressedasunitspergram.

11. 2. Pigeon Method
Minimum6pigeonsareusedfortestingeachsample.Theweightoftheheaviestpigeon
shouldnotexceedtwicetheweightofthelightestpigeon.Foodiswithdraw16-28hrs
beforetheexperiment.
Pigeonsaredividedonthebasisoftheirsex,weightandbreedinto2gps.Theyare
anaesthetizedwithanestheticether.Onesideofthewingisdissected&thealarveinis
cannulatedbymeansofavenouscannula.Dilutionsaremadewithnormalsaline.
Testsample&STDsampleisinfusedthroughcannula.Inpigeons,stoppageofheartis
associatedwithacharacteristicvomitingresponsecalled‘emesis’.
Themilkfromthecropsacofpigeonsisbeingejectedout.Thismaybetakenastheend
pointresponseofdigitalis.

12. Thelethaldoseperkgofbodyweightisdeterminedforeachpigeon.
Potencyofthetestsample=MeanlethaldoseofSTD/Meanlethaldoseofthetest.

13. BIOASSAY OF d-TUBOCURARINE

14. BIOASSAY OF d-TUBOCURARINE
1.RabbitHead-dropMethod:
Principle:
d-TubocararineHClisinjectedintothemarginalveinofarabbit’seartillthe
rabbit’sneckmusclesarerelaxedsuchthattheanimalcannotholditshead
up.Thetotalamountoftestsamplerequiredtoproducetheendpointis
comparedwiththetotalamountoftheSTDsamplerequiredtoproduce
similarendpoint.
SelectionofRabbits:
Rabbitsweighing2kgareused.Animalsshouldbefreefromdisease,
obtainedfromahealthycolonyandshouldbeaccustomedwiththe
experimentalprocedure.

15. BIOASSAY OF d-TUBOCURARINE
ExperimentalProcedure:
Rabbitisplacedinaholderwithitsheadprotrudingoutside.Thehead
shouldbefreelymovable.
Minimum8rabbitsareused.Theyaredividedinto2groupseach
containing4rabbits.
FirstgroupwillreceiveSTDsampleandthesecondgroupwillreceivethe
sampleundertestd-Tubocurarinesolutionisinjectedataconstantspeed
byinfusionapparatusthroughthemarginalvein.
Injectionshouldbegivenatarateof0.4ml/minandshouldtakeabout10
min.Dose0.012%w/vinsaline.

16. BIOASSAY OF d-TUBOCURARINE
Infusioniscontinuedtilltherabbitwillnotbeinapositiontoholdits
headerectortherewillbenoresponsebyfocusinglightontheeyes.
Rabbitsrecoverimmediatelyfromtheeffectofcurarization.
Duringtheexperimentthereisapossibilityofrespiratoryembarrassment
whichistreatedbyinjectingNeostigminemethylsulphate(0.05mg.)and
atropinesulphateimmediatelythroughthemarginalearvein.
Cross-overtestiscarriedouttominimizebiologicalerrorduetoanimal
variation.
ThoserabbitswhichreceivedtheSTDsampleonthefirstdaywillbe
giventestsampleontheseconddayofexperimentandviceversa.
Meandosewhichproducesheaddropofthetestsampleiscompared
withthemeandoseofstandardpreparation.

17. BIOASSAY OF d-TUBOCURARINE
2.Frogs Rectus Abdominusmuscle Preparation:
Afrogispithedandlaidonitsbackonacorkcoveredboardtowhichitis
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.

18. BIOASSAY OF d-TUBOCURARINE
Frogs Rectus Abdominismuscle Preparation:
Theresponsesarerecordedusingisotonicfrontalwritingleverwith1g
tension.
TwosimilarcontractionswiththesameconcentrationofAchare
obtained.
ThreedosesoftheSTDsampleandoneintermediatedoseofthetest
sampleareselected&thereductioninheightofcontractioninducedby
Achisnoteddown.
Achcontractionisrecordedonslowmovingdrumfor90sec.d-
Tubocurarineisallowedtoactfor30sec.
Thepercentagereductionateachdoselevelsiscalculatedandlogdose
responsecurveoftheSTDdrugisplotted.Alinearresponsewillbe
obtained.Thepotencyoftestsampleiscalculatedfromthestandard
curve.

19. BIOASSAY OF HISTAMINE

20. BIOASSAY OF HISTAMINE
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.
Bioassayofhistamineonisolatedguineapigileumcanbedeterminedbygraded
bioassayprocedurei.e.i)Matchingbioassay,ii)Interpolationbioassay,iii)
Bracketingassay,iv)Multiplepointassays.

21. Bioassayofhistamineinbiologicalsamplescanbestudiedbyusingdifferent
bioassaymethods.Dependinguponpharmacologicalaction,histaminecanbe
assayedby:
Contractile effect of isolated ileum of guinea pig,
Contractile effect of uterus of guinea pig, and
Fall in blood pressure of anaesthetized and atropinizedcat
Bioassay of histamine using guinea pig ileum:
Principle
Guinea pig ileum is very useful preparation for the bioassay methods. It is more
sensitive to histamine. Contractile response of histamine to ileum is due to
presence of H1 receptor.

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

23. Tracetheileocaecaljunctionbyliftingthecaecum,thengoupwardsupto8cm
fromthejunctionandcutthe2-3cmlongsegmentofileummuscle(excluding
theterminal5-8cm,containsexcessofexcitatoryα-adrenergicreceptor,which
mayinterfereinthestudy).
ImmediatelyplaceitinapetridishcontainingaeratedwarmTyrodesolution.
Slowlyremovethemesenteryattachedtothemuscle&thengentlycleanthe
lumenofileumbypassinglukewarmTyrodesolutionthroughitwithapipette
orsyringe.
MountthepreparationintheinnerorganbathcontainingTyrodesolution(20
ml)maintainedat37ºC.Tiethebottomendofthemuscletothehookof
aerationtubeandtheupperendtotheisotonicfrontalleverbyathread
withoutclosingthelumen.

24. Adjustthemagnificationofresponseto7-10fold.AeratethetissuewithO2
orcarbogenslowly(40-60bubblesperminutes).
Stabilizethetissuefor30minbyapplyingatensionof0.5gweightattached
tothelever,duringwhichwashthetissuewithfreshTyrodesolutiononcein
every10min.
Use5mintimecyclewithcontacttimeof30secforrecordingthecontraction
dependentresponsesoftissueduetohistamineonthesmokeddrums.
RecordtheresponsesofSTD&testcompounds.
Recordtheresponsesoftestcompoundi.e.unknownconcentrationof
histaminewithgraduallyincreasingvolume,tillobtainingaresponse(T)which
wouldlieonthelinearportion(30-70%)oftheCRC.Fixtheresponseobtained
duetovolumeofT.

25. RecordthegradedresponsesofSTDsolution&testsolutionofhistamine.
Thepotencyofthetestsampleiscalculatedinrelationtothatofthestd.
preparationbydividingtheaveragelethaldoseofthesampletothetestand
expressedasunitspergram.
Potency=LethaldoseofSTD/Lethaldoseoftest=unit/g.

26. BIOASSAY OF 5-HT

27. 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.Isolatedfundusstripofratstomach,
2.Isolatedterminalcolonofrat,
3.Isolatedratuterus&4.Perfusedrabbitear.

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

29. British Journal of Pharmacology, Volume: 120, Issue: S1, Pages: 142-147, First
published: 08 September 2011, DOI: (10.1111/j.1476-5381.1997.tb06791.x)

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

32. MountthepreparationintheinnerorganbathcontainingKrebssolution(20
ml)maintainedat37ºC.Tiethebottomendofthemuscletothehookof
aerationtube&theupperendtotheisotonicfrontalleverbythread.Adjust
themagnificationofresponseto10-15fold.AeratethetissuewithO2or
carbogenslowly(40-60bubblesperminutes).
Apply1gload&stabilizethetissuefor30minduringwhichwashthetissue
withfreshKrebssolutiononceinevery10min.
Usea5mintimecyclewithcontacttimeof90s(sincethefundusstripmuscle
contractsslowlyandrelaxesslowly)forrecordingthecontractiondueto
serotonin.
RecordthegradedresponsesofSTDsolution&testsolutionofserotonin.
Thepotencyofthetestsampleiscalculatedinrelationtothatofthestd.
preparationbydividingtheaveragelethaldoseofthesampletothetestand
expressedasunitspergram.

33. BIOASSAY OF ACTH

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

35. Packing: Preserve in single-dose or multiple-dose containers of Type-1 glass.
Storage: Store in cold place.
Labeling: Injection recommends intravenous administration.
Purpose and rationale:
Thisisahistoricalassaymethod.AdministrationofpituitaryACTHdecrease
theascorbicacidpresentintheadrenals.Thedepletionofadrenalascorbic
acidisafunctionofthedoseofACTHadministered.Thisrelationship
hasbeenusedforaquantitativeassayofACTH.

36. Solutions:
SolutionA:Fiveunitsoftestorstandarddissolvedin0.25mlof0.5%phenol
solutionanddilutedwith8.1mlof15%gelatinsolution(Now0.5mlcontain
300mUACTH).
SolutionB:ThreemlofsolutionA dilutedwith6 ml
gelatinsolution.Nowconcentrationreducedto100mUACTH/0.5ml.
SolutionC:Again3mlofsolutionBdilutedwith6mlofgelatinsolution,the
resultingsolutioncontains33mUACTH/0.5ml.

37. Procedure
MaleWistarrat(100-200g)arehypophysectomized(pituitarygland
removedbysurgery)onedaypriortothetest.
Foronetestwith3doseofTestpreparationandSTDsolutionusedforthe
study.
Numberofhypophysectomizedratsrequired:atleast36(preferably
60).
Thehypophysectomizedratsarerandomlydistributedintosixgroups.Each
ratreceivessubcutaneous0.5mlofthevariousconcentrationsoftestor
standard.

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

39. Ascorbic acid determination:
Reagents
0.02% ascorbic acid solution
85 % sulfuric acid (9N H2SO4)
0.02 g/ml of dinitrophenolhydrazine in 9N H2SO4
0.06 g/ml of thioureaare dissolved in distilled water
Charcoal
Preparationof0.02%ascorbicacidsolution
100mgL-ascorbicacidaredissolvedin100mlof4%trichloroaceticacid
(1mg/mlsolution)(SolutionA=1%solution)
2mlofSolutionAdilutedin10mlof4%trichloroaceticacidtoachievea
0.2%ascorbicacidsolution(solutionB)
1mlofsolutionBdilutedin10mlof4%trichloroaceticacidtoachievea
0.02%ascorbicacidsolution(solutionC)

40. Preparationofothersolutions
Sulfuricacid(85%)isobtainedbyadding900mlconcentratedsulfuric
acidto100mldistilledwater.
Twogmdinitrophenolhydrazinearedissolvedin100ml9NH
2SO
4(75ml
distilledwater&25mlconcentratedsulfuricacid).
Sixgmthioureaaredissolvedin100mldistilledwater.
Calibration
Trichloroaceticacid(4%)isaddedto0.0,0.5,1.0,2.0,3.0,4.0,6.0,8.0ml
ofthe0.02%ascorbicacidsolution(solutionC)and1.0,1,5and2.0mlof
the0.2%ascorbicacidsolutiontoreachafinalvolumeof8.0ml(Solution
B).
100mgcharcoalisaddedtoeachsampleandthoroughlymixedby
shakingfor1min.
After5minthesolutionsarefiltered

41. Ascorbic acid determination:
Analiquotof0.1mlofthe6%thioureasolutionisaddedto2.0mlofthe
filtratefollowedby0.5mldinitrophenylhydrazinesolution.
Themixtureisshakenandheatedfor45minat57°Cinawaterbath.
Thesolutionsareplacedinanice-coldwaterbathandwithfurthercooling
2.5mlofthe85%sulfuricacidareadded.
Thecalibrationcurveisestablishedatawavelengthof540nmusingthe
solutionswithoutascorbicacidasblank.