b1. Bioassay of ACTH.pdf

Bioassay of ACTH/ Adrenocorticotropic Hormone/
corticotropin

1. BioassayofACTH/
AdrenocorticotropicHormone/
corticotropin
Mr. Vishal Balakrushna Jadhav
Assistant Professor (Pharmacology)
School of Pharmaceutical Sciences (SOPS)
Sandip University, Nashik
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2. AboutACTH
ACTH(corticotropin)ispolypeptidetropic
hormone(39aminoacids)secretedbythe
anteriorpituitarygland.
ACTHstimulatestheproductionofcortisol,
asteroidhormoneimportantforregulating
glucose,proteinandlipidmetabolism,
suppressingtheimmunesystemresponse,
andhelpingtomaintainbloodpressure.
Regulationofcortisolsynthesis
Whencortisollevelfalls,thehypothalamus
producescorticotropinreleasinghormone
(CRH)→CRHstimulateshypothalamusto
releaseofACTH→ACTHthenstimulatesthe
adrenalgland(asshowninfigure).
2

3. 1)Addisonsyndrome
Characterizedbyprimaryadrenal
insufficiency→↓cortisolproduction
duetoadrenalglanddamage,and
secondaryadrenalinsufficiency→
decreasedcortisolproductionbecause
ofpituitarydysfunction.
Alsoknownasprimaryadrenal
insufficiencyandhypocortisolism,isa
long-termendocrinedisorderinwhich
theadrenalglandsdonotproduce
enoughsteroidhormones.Symptoms
generallycomeonslowlyandmay
includeabdominalpain,weakness,and
weightloss.
Whoneedexternalcortisol?

4. 2)Hypopituitarism
Alsocalledpituitaryinsufficiency,isarareconditioninwhichyourpituitary
glanddoesn'tmakeenoughofcertainhormones.Symptomscanincludeoneor
moreofthefollowing:
Stomachpain,decreasedappetite,nauseaandvomiting,constipation,
Excessivethirstandurination,
Fatigueand/orweakness,
Anemia(nothavingenoughredbloodcells),
Headacheanddizziness,
Sensitivitytocold,
Weightlossorweightgain,
Musclesaches,
Inwomen:lossofarmpitorpubichair,decreasedsexdrive,infertility,
problemswithbreastfeeding,irregularornomenstrualperiods,
Inmen:lossofhair(ontheface,orinthearmpitsorpubicarea),decreased
sexdrive,infertility,erectiledysfunction,and
Inchildren,problemswithgrowth(includingheight)andsexualdevelopment.

5. ACTHOfficialPreparations
Corticotropininjectionisasterilesolution,inasuitablediluent,of
thepolypeptidefromthepituitaryglandsofmammals.Potency
rangeshouldbe80.0–120.0%ofUSPcartiotropinunits.
Corticotropinforinjection,antimicrobialagent.
Repositorycorticotropininjectioniscorticotropininasterile
solutionofpartiallyhydrolyzedgelatinandisintendedfor
subcutaneousandintramuscularuse.Thissolutionhasbeen
adoptedasthereferencestandardforthebioassay.
PackingPreserveinsingle-doseormultiple-dosecontainersof
Type-1glass.
StorageStoreincoldplace.
LabelingInjectionrecommendsintravenousadministration.
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6. Bioassay ofACTH
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1) AdrenalAscorbicAcidDepletion
 Purposeandrationale
Thisisnowahistoricalassay,whichhoweverhasbeenusedextensivelyfor
standardizationofACTHpreparations.
Theadministrationofpituitaryadrenocorticotropichormone(ACTH)is
followedbyadecreaseintheamountofascorbicacidpresentinthe
adrenals.
ThedepletionofadrenalascorbicacidisafunctionofthedoseofACTH
administered.
ThisrelationshipwasusedforaquantitativeassayofACTHbySayersetal.
(1948).Furthermore,thetesthasbeenusedforevaluationofsynthetic
corticotropinanalogs.
Asimilartestisusedforluteinizinghormoneactionintheratovary.

7.  Solution
FiveunitsoftheInternationalStandardforcorticotropin(Banghametal.
1962)oranamountoftestpreparationsupposedtocontainabout5unitsare
dissolvedin0.25mlof0.5%phenolsolutionanddilutedwith8.1mlof15%
gelatinsolution.Inthisway,0.5mlcontains300mUACTH.
Then3mlofthissolutionisdilutedwith6.0mlgelatinsolution(toprevent
adsorptiontoglassware),resultingin100mUACTHper0.5ml.
Then3mlofthissolutionisagaindilutedwith6.0mlgelatinsolution,resulting
inacontentof33mUACTHper0.5ml.
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8.  Procedure
MaleWistarratsweighingbetween100and200garehypophysectomized1day
priortothetest.Therangeofweightsinanyonetestshouldnotexceed15g.
Foronetestwiththreedosesoftestpreparationandstandard,atleast36,
preferably60,hypophysectomizedratsarenecessary.
Thehypophysectomizedratsarerandomlydistributedtosixgroups.
Eachratreceivessubcutaneously0.5mlofoneofthevariousconcentrationsof
testpreparationorstandard.
Threehoursafterinjection,theanimalsareanesthetized,bothadrenals
removed,
freedfromextraneoustissueandweighed.
Theratsaresacrificedandtheskullopenedtoverifycompletenessof
hypophysectomy.
Theadrenalsarehomogenizedin4%trichloroaceticacidandtheascorbicacid
determinedaccordingtothemethodofRoeandKuether(1943).
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9. Hypophysis or pituitarygland
Hypophysectomyis the surgical removal of thehypophysis. 9

10.  Ascorbic aciddetermination
 Reagents
First100mgL-ascorbicacidisdissolvedin100ml4%trichloroaceticacid
and20mlofthissolutionisdilutedwith4%trichloroaceticacidtoachieve
a0.2%ascorbicacidsolution;2mlofthissolutionisdilutedwith4%
trichloroaceticacidtoachievea0.02%ascorbicacidsolution.
Sulfuricacid(85%)isobtainedbyadding900mlconcentratedsulfuric
acidto100mldistilledwater.
Then2gdinitrophenylhydrazine(DNPH)isdissolvedin100mlof9N
H
2SO
4(75mldistilledwaterand25mlconcentratedsulfuricacid),and
6gthioureaisdissolvedin100mldistilledwater.
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11.  Calibration
Trichloroaceticacid(4%)isaddedto0.0,0.5,1.0,2.0,3.0,4.0,6.0,and8.0
mlofthe0.02%ascorbicacidsolutionand1.0,1.5,and2.0mlofthe0.2%
ascorbicacidsolutiontoreachafinalvolumeof8.0ml.
Then100mgcharcoalisaddedtoeachsampleandthoroughlymixedby
shakingfor1min.
After5minthesolutionsarefiltered.
Analiquotof0.1mlofthe6%thioureasolutionisaddedto2.0mlofthe
filtratefollowedby0.5mldinitrophenylhydrazinesolution.
Themixtureisshakenandheatedfor45minat57°Cinawaterbath.
Thesolutionsareplacedinanice-coldwaterbathandwithfurther
cooling2.5mlofthe85%sulfuricacidisadded.
Thecalibrationcurveisestablishedatawavelengthof540μmusingthe
solutionswithoutascorbicacidasblank. 11

12.  Preparationoftheadrenals
Bothadrenalsarehomogenizedinglasstubescontaining200mg
purifiedsandand8.0mlof4%trichloroaceticacid.Thereagentsare
addedasdescribedforthecalibrationcurve.
 Evaluation
Thepotencyratioincludingconfidencelimitsiscalculatedwiththe3+3-
pointassay(threepointbioassay,latinsquaredesignshownbelow)by
usingmeanconcentrationsofstandardandtest.
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S1 S2 S3 T1 T2 T3
S2 S3 T1 T2 T3 S1
S3 T1 T2 T3 S1 S2
T1 T2 T3 S1 S2 S3
T2 T3 S1 S2 S3 T1
T3 S1 S2 S3 T1 T2

13. Flowchart indicating bioassay of ACTH
Numberofsamples:3standard+3test
Numberofanimals:minimum36
(6/group)(preferably60-10/group)
Onedaypriortothetest,pituitary
glandremovedbysurgery
Administer0.5ml
ofthevarious
conc.ofstandard
Administer0.5ml
ofthevarious
conc.oftest
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14. sacrificedandthe
scullopenedto
verifycompleteness
ofhypophysectomy
Theadrenalsarehomogenizedinglasstubes
contains200mgpuresandand8.0mlof4%
trichloroaceticacidandtheascorbicacid
determined.(RoeandKuether1943).
3hrafterinj.animalsareanesthetizedandboth
adrenalsremovedforascorbicacidestimation
Administer0.5mlofthe
variousconc.ofSTD
Administer0.5mlofthe
variousconc.oftest
Potencyratiocalculatedby3+3pointassay
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15. Flowchart indicating estimation ofascorbicacid
Prepare1mg/mlconc.of
ascorbicacidin4%TCA(stock)
SolutionA
Use solution A to prepare 0.2% of
ascorbic acid in 4% TCA (SolutionB)
Use solution B to prepare 0.02% of
ascorbic acid in 4% TCA (SolutionB)
Preparation of calibration curve usingstandard
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16. 2)CorticosteroneBloodLevelsinDexamethasone-BlockedRats
 Purposeandrationale
Corticotropinactivitycanbemeasuredbytheincreaseofcorticosteronein
venousbloodofhypophysectomizedrats(nolongerrequired)or
dexamethasone-blockedrats.
Thetestcanbeusedtomeasuretime-responsecurvesofcorticotropin
analogsordepotpreparations.
Thesensitivitycanbeincreasedbydeterminingcorticosteroneinadrenal
venousbloodaftercannulationoftheadrenalvein.
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17. 17
 Procedure
MaleSprague-Dawleyratsweighing150–200areinjectedsubcutaneously
24hand1hpriortosubcutaneousinjectionoftheACTHpreparationor
thestandardwith5mg/kgdexamethasoneinoilysolution.
Eightratsareusedforeachdoseoftestpreparationorstandard.
AtincreasingtimeintervalsafterACTHinjection,theratsareanesthetized
with60mg/kgpentobarbitali.p.andbloodiswithdrawnbyvenipuncture
(alternativelybyretro-orbitalpuncture).
Next,1mlplasmaisdilutedwith2mldistilledwaterandextracted
(washed)with5mlpetrolethertoremovethelipids.Thepetroletheris
discardedand2mlofthewaterlayerisextractedtwicewith5ml
methylenechloridebyvigorousshakingfor15min.
Themethylenechloridephaseisseparatedbycentrifugation.
Bothmethylenechlorideextractsareunifiedandshakenwith1mlice-cold
0.1NNaOH.

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Thewaterphaseisimmediatelyremovedandthemethylenechloride
extractsdriedbyadditionofdrysodiumsulfate.
A5mlaliquotofthemethylenechlorideextractismixedwith5mlofthe
fluorescencereagent(7partsconcentratedsulfuricacid,3parts96%
ethanol,v/v).
Aftervigorousshaking,themethylenechloridephaseisremovedand
fluorescenceismeasuredwithprimaryfiltersof436μmandsecondary
filtersof530–545μm.
Forcalibration,concentrationsof0,20,50,100,and250μg/ml
corticosteronearetreatedidenticallyandmeasuredineachassay.
 Evaluation
Usingthreedosesoftestcompoundandstandard,potencyratioswith
confidencelimitscanbedeterminedforeachtimeintervalwiththe3+3-
pointassaygivingevidenceforthedurationofaction.

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