BIO ASSAY OF OXYTOCIN

J.S.K. NAGARAJAN

ASST. PROF.,

JSS UNIVERSITY,

(OFF CAMPUS: JSS COLLEGE OF PHARMACY,

OOTACAMUND – 643001

Ph: 94431 49945

Email: nagasaki2001@rediffmail.com

OXYTOCIN• OXY – RAPID TOCOS- LABOR• Synthesized in both sexes, well recognized physiological effects

only in women.• Cyclic Polypeptide hormone - from posterior pituitary gland.• Pituitary gland consist posterior labe which produce oxytocin

and diuretic hormone.• Neurosecretary product mainly synthesize in the cell bodies of

paraventracular nuclear of the hypothalamus.

ROLE OF OXYTOCIN:• Stimulate the contraction of the uterine smooth muscle &

memory gland.• Oestrogen progesterone & prolactin – responsible for production

of milk by memory gland but milk ejection require oxytocin.• Facilitates the contraction of uterus.

It is presented as a solid or solution in a solvent containing an appropriate antimicrobial preservative such as 0.2% w/v of chlorbutol.

Animal species - 90 - 110% stated number of units of oxytocin activity.

Synthetic: Solid - NLT 560 units/mg Calculated with reference to the peptide content & when liquid NLT 150 units/ml

Oxytocin – Mechanism of action Neuropeptide made in hypothalamus that stimulates

contractions that expel the infant from uterus.

Responsible for milk letdown & triggered by the nipple stimulation of suckling

Called love & bonding hormone. It has a very special affect on mothering .

Psychologically, oxytocin promotes a feeling of well being and tranquility.

It enables the growing sense of love and attachment to the infant. The more the infant suck the more oxytocin is produced.

In mothers it increases their attachment to their infant, promoting the feeling of love, and makes her infant more valuable to her.

It also suppresses the fear that would normally cause her to back off from threat.

BIOLOGICAL ASSAY OF OXYTOCIN:

PRINCIPLE:

Potency is determined by comparing its activity – Depression of BP– Contraction of Uterus– Milk Ejection Pressure– Vasopressor activity

with standard preparation of oxytocin

STANDARD PREPARATION:

Consisting free dried synthetic oxytocin peptide with human albumin citric acid (12.5 units)

METHOD-A (Depression of the BP in chicken)

Test Animals: Cockerel (young male chicken), 1.2 - 2.3 Kg, Healthy

Anaesthesised cock-prolonged & constant high B.P

Expose gluteus primus muscle(thigh) & remove politeal artery & crural vein.

Cannulate the popliteal artery & record B.P response

Cannulate the crural or brachial vein.

Prepare std soln with saline. Inject 0.1 - 0.5ml

Inject 2 doses of std soln into cannulate vein is record B.P response

Dose should cause decrease in B.P (reqd. dose between 20-100mUnits)

Interval bween 2 injection, bween 3-10mins depend on rate @ which B.P return normal

Dil. test preparation with saline so as to get same response as standard

The ratio between standard & test should be equal

If animal rapidly becomes insensitive to repeated injection the soln another must used.

Measure all responses are calculated result of the assay by std statistical method.

METHOD—B: (By contraction of the rat uterus):Test animals: Female rat 120 – 200g

Inject 100ug of oestradiol benzoate IM into female rat before the assay

Immediately before assay confirm by vaginal smear that rate in oestrus or pre oestrus.

Kill rat & suspend one horn of uterus in organ bath containing a solution of following Nacl,Kcl,Cacl2, NaHco3, Na2Hpo4, NaH2po4, Mgcl2, Dextrose

Maintain the bath at temp at of 32 c

Bath liquid required dose between 10-50 units/ml.

Oxygenate solution with mix of 95% of O2, 5% of CO2 record -contraction of muscle.

Record contraction produces by addition of two dose of std. ppn (Reqd. Dose 10 & 50munits/ml of bath liquid)

when maximum contraction has been reached replace - bath liquid by fresh solution.

Dose should be added at regular interval[3-5minutes]

Similarly record the contraction of test preparation as standard.

Ratio between two dose of test & two dose of std should be equal. This ratio kept constant through out the assay.

Measure all response & calculate result of assay by standard statistical method.

METHOD C: (Milk ejection pressure in Lactating rat)TEST ANIMALS: Lactating rat, 3-21 day after parturition, 300 g Separate from litter & 30-60 minutes later anaesthetise (IP Pentobarbitone

Na).

Tie rat to an operating table, at 37º, by its hind legs leaving front legs free.

Cannulate trachea with a short PE tube of i.d. 2.5 mm in such a manner so as to ensure a free airway; apply artificial respiration only if necessary.

Cannulate an external jugular or femoral vein with a PE tube of i.d. 0.4 mm filled with saline & closed with a pin.

Shave the skin surrounding the inguinal and abdominal teats and excise the tip of one teat, preferably the lower inguinal teat.

Insert a PE tube of i.d. 0.3 mm & e.d. 0.6 mm, to a depth sufficient to obtain appropriate measurement of pressure (3-10 mm depth), into the

primary teat duct which opens onto the cut surface and tie firmly in place with a ligature.

Connect this cannula with a suitable strain gauge transducer (such as that used for recording arterial BP in rat) and fill with a 3.8% w/v of Na citrate

/saline contain 50 Units of heparin Na/ml to prevent clotting of milk.

After cannulation, inject 0.05 - 0.2 ml of this solution into teat duct through transducer to clear milk from tip of the cannula. (This procedure may be repeated during the assay should obstruction arise from milk ejected into

the cannula).

Clamp the strain gauge so that a slight tension is applied to the teat and its natural alignment is preserved and connect the gauge to a potentiometric

recorder adjusted to give full-scale deflection for an increase in milk-ejection pressure of 5.3 kPa.

Inject all solutions through the venous cannula using a 1-ml syringe graduated in 0.01 ml and wash them in with 0.2 ml of saline.

Prepare a solution of Std. & Test Ppn in saline solution so that the volume to be injected is between 0.1 - 0.4 ml.

Choose two doses of Std Ppn such that the increase in milk-ejection pressure is about 1.35 kPa for Lr dose and about 2.7 kPa for Hr dose.

As an initial approximation, a lower dose of between 0.1 and 0.4 milliUnit and an upper dose of 1.5 to 2 times this amount may be tried.

Choose two doses of the Test Ppn with the same inter-dose ratio, matching effects of doses of the Std Ppn as closely as possible.

Inject four doses (2 doses of Std & 2 doses of Test) at intervals of 3- 5 minutes.

2 doses of Std and 2 doses of test should be given according to randomised block or a Latin square design & at least four responses to each -recorded.

Measure all responses & calculate result of the assay by std statistical methods.

Potency - 90% - 111%. Fiducial limits of error are 80% - 125%stated potency.

Vasopressor activity: NMT 0.5 Unit /20 Units of oxytocic activity - by biological assay for vasopressor activity- comparing activity of Test & Std Ppn of arginine vasopressinFreeze-dried syn. arginine vasopressin peptide acetate with human albumin & citric acid

(supplied in ampoules containing 8.20 Units)

Inject slowly into tail vein of male albino rat weighing 300g -solution of a suitable a-adrenoceptor blocking agent, (10 ml/kg body weight of solution prepared by dissolving 5 mg of phenoxybenzamine HCl in 0.1 ml of ethanol (95%), adding 0.05 ml of 1 M HCl &

dil to 5ml with saline .

After 18 hours, anaesthetise rat - that will maintain -prolonged & uniform BP.

After 45-60 minutes, tie the rat on its back to the operating table by its hind legs.

Cannulate trachea with short PE of E.D. 2.5 mm & dissect carotid artery ready for cannulation.

Then cannulate the femoral vein close to the inguinal ligament.

Retract the abdominal muscles to expose the inguinal ligament.

Retract superficial pudendal vein to one side & dissect femoral vein towards inguinal ligament from corresponding artery.

When dissecting, a deep branch reaching femoral vein must be found & tied off to prevent bleeding during cannulation.

Tie a short PE cannula of E.D. about 1 mm into femoral vein by two ligatures & join by a short piece of flexible tubing to a 1-ml burette with an attached thistle funnel containing

saline at about 37º.

Firmly fix wet absorbent cotton swab to thigh so as to cover incision and cannula. At this stage inject through venous cannula 200 Units of heparin, dissolved in saline /100 g

of body weight.

Then tie in a carotid cannula of E.D.about 1 mm & connect by a column of saline contain heparin with a pressure measuring device such as Hg

manometer of I.D. about 2-3 mm.

central & peripheral nervous system including both vagus & associated sympathetic nerves is left intact.

No artificial respiration is necessary.

No air is injected, inject all solutions through venous cannula by means of a 1-ml syringe & wash in with 0.2 ml of saline from burette.

Dil extract of Std & Test Ppn with saline so that volume to be injected is between 0.1 & 0.5 ml.

Choose 2 doses of the Std Ppn such that the elevation of the BP is about 4 kPa for Lr dose & about 7 kPa but always submaximal for higher, ratio of low to high dose being determined by response & usually being 3-5.

As an initial approximation doses of 3 and 5 MUnits may be tried.

Choose 2 doses of Test ppn with same inter-dose ratio, matching effects of dose of Std Ppn.

Inject doses at intervals of 10 - 15 minutes.

2 doses of Std & 2 doses of Test Ppn should given in randomised block / Latin square design & 4-5 responses to each recorded.

Measure all responses & calculate result of the assay by Std statistical methods.

METHOD A METHOD B METHOD C Method DDepression of BP Contraction of Uterus Milk ejection Pr.in Lactating

ratVasopressor Activity

animal

Cockerel, 1.2 to 2.3 Kg Female rat 120 200g Lactating Rat,300g 3-21 days parturitiction.

Male rat 300g

Organ Anaesthetised , prolonged & const high BP

Gluteus Primus muscle (thigh) & remove politeal artery & crural vein.Cannulate Popliteal artery & record BP.

Inject 100ug oestradiol by IM. Confirm rat in oestrus /pre oestrus by vaginal smear.Kill rat & suspend one horn of uterus in organ bath contain Na, K, Ca, Mg chrloride NaHCO3,NaHPO4 and dextrose- bath 32c

anaesthetise(Pentobarbitone Na IP). Tie hind legs leaving front legs free,@37c. Cannulate trachea-respirate artificially if reqd. Shave skin around inguinal abdominal teats . Pr. 3-10mm. Connect cannula with gauge transducer & fill with 3.8% Na citrate/saline contain 50 Units heparin Na/ml to prevent clotting of milk. After cannulation, inject 0.05 - 0.2 ml of this solution into teat duct through transducer to clear milk from tip of cannula. Clamp strain gauge so that a slight tension is applied to teat & its natural alignment is preserved & connect gauge to a potentiometric recorder adjusted to -for increase milk-ejection pressure of 5.3 kPa.

2 doses of Std Ppn - increase in milk-ejection pressure is 1.35 kPa for Lr dose & 2.7kPa - Hr dose.

Inject adrenoceptor blocking agent (phenoxybenzamine 5mg) into tail vein of rat, after 18hrs, anaesthetise rat-prolonged & uniform BP. After 45-60mints, tie rat on its back to operating table by its hind legs. Cannulate trachea with PE & dissect carotid artery ready for cannulation. Cannulate femoral vein close to inguinal ligament. Retract superficial pudendal vein to one side & dissect femoral vein towards inguinal ligament from corresponding artery. When dissecting, a deep branch reaching femoral vein must be found & tied off to prevent bleeding. Tie a PE into femoral vein by two ligatures & join by a short piece of flexible tubing to burette with attached thistle funnel containing saline-37º.Inject 200Units of heparin, dissolved in saline/100g of body weight thru venous cannula. Tie in a carotid cannula & connect by column of saline contain heparin with pressure measuring device-CNS & PNS including both vagus & associated sympathetic nerves is left intact. No air is injected, inject all solutions thru venous cannula by means of syringe & wash with 0.2 ml of saline

Std With Saline 0.1 -0.5ml 10-50units/ml 0.1-04 ml 0.1 & 0.5ml

Inject 2 doses of std solution into cannulate vein record BP

Oxygenate the soln. with 95% O2, 5%CO2 record the contraction.

2 doses of the Test Ppn with the same inter-dose ratio, matching effects of doses of the Std Ppn as closely as possible.

2 doses Std Ppn -elevation BP 4 kPa for Lr & 7 kPa higher dose, ratio of low to high dose being determined using 3-5 munits

Decrease of BP Record the contraction by addition of 2 doses of STD.

Interval

3- 10 mins bween 2 injection- depend on rate of BP returns normal

3-5 minutes 3-5 mints Inject doses at intervals of 10 - 15 minutes.

Test Dil. Test ppn with saline get same response

As per std. 2 doses Std & 2 doses test - randomised block/Latin square design & at least 4 responses to each -recorded.

2 doses of std & Test randomised block/Latin square design & 4-5 response recorded.

ratio Between Standard & Test equal