Oxytocin bioassay

1. Oxytocin bioassay
Dr. S. Parasuraman, M.Pharm., Ph.D.,
Associate Professor & Unit Head,
Unit of Pharmacology,
Faculty of Pharmacy, AIMST University,
Bedong 08100, Malaysia.

2. Oxytocin
• Oxytocin is a peptide hormone and neuropeptide, produced in
the hypothalamus and released into the bloodstream by the
pituitary gland. Oxytocin is an essential hormone for childbirth
(facilitate childbirth) and lactation.
• Functions:
• Oxytocin stimulates the muscles of the uterus to contract, and boosts
the production of prostaglandins, which also increase uterine
contractions.
• Once your baby is born, oxytocin promotes lactation by causing
contractions of the myoepithelial cells in the alveolar ducts of your
breasts.

3. Oxytocin Bioassay
• Purpose:
• Bioassays provide information concerning the potency of
biological products. This is essential for evaluating batch-to-
batch consistency and stability.
• Bioassay data are crucial at all stages in the development of
biological products, from early research work to final quality
control of finished products.

4. Oxytocin Bioassay
• Methods:
• Isolated uterus
• Chicken blood pressure
• Milk ejection in the lactating rabbit or rat

5. Oxytocin Bioassay - Isolated uterus
Requirements:
• Animal model: Female Sprague-Dawley or Wistar rats
• Age: 120–200 g
• Tissue preparation: Isolated rat uterus
• Physiological salt solution: de Jalon solution
• The bath temperature: 32 °C
• Aeration: mixture of 95% O2 and 5% CO2
• Standard preparation rang: 10 - 50 microunits per ml organ
bath
• Kymograph and Kymograph drum with frontal writing lever/
auxotonic lever or Student digital Physiograph or Biopac/
AD data acquisition system

6. Oxytocin Bioassay - Isolated uterus
Procedure:
• 18 - 20 h prior to the assay, the rat is injected i.m. with 100
μg of estradiol benzoate for priming (receptor induction).
• Before the assay they will be tested for estrogen induced
epithelial proliferation by vaginal smear test (immediately
before the assay confirm rats is in oestrus or proestrus
stage).
Stages of estrous cycle

7. Oxytocin Bioassay - Isolated uterus
Procedure:
• Rat will be anathematized, and
hysterectomy is performed to remove the
one horn of the uterus and is suspended
in an organ bath containing a physiological
salt solution, maintained at 32 °C and
aerated with mixture of 95% O2 and 5%
CO2.
• The preparation is loaded with 1–2 g and
the contractions (DRC) recorded
Kymograph/ Physiograph.
• Unknown concentrations will be
determined by using either interpolation/
multiple point bioassay.

8. Oxytocin Bioassay - Isolated uterus

9. Oxytocin Bioassay - Chicken blood pressure
Principle:
• Oxytocin induces a transient fall in blood pressure in
chicken and other birds. This effect can be used as assay for
oxytocin.
Procedure:
• White Leghorn chickens weighing 1.2 to 2.0 kg are
anesthetized by intravenous injection with 200 mg/kg
sodium phenobarbital via the brachial vein.
• The ischiadic artery/ popliteal artery, crural vein are
exposed by removing the feathers from the outer surface (7
- 8 cm).
• Cannulate the artery for blood pressure monitoring and
crural vein for infusion of test or standard oxytocin.

10. Oxytocin Bioassay - Chicken blood pressure
Procedure (Cont.,):
• The interval between injections should be constant ( 3 to 10
mins). The ratio between the standard and test should be
equal and should be constant throughout the assay.
• Observation:
• Intravenous injection of oxytocin induces in chickens an
immediate, transient fall in blood pressure. The required
doses normally lie between 20 and 100 mU.
• The responses of each dose are averaged and potency
ratios with confidence limits calculated from the 2 + 2-
points assay.

11. Oxytocin Bioassay - Milk ejection in the
lactating rabbit or rat
• This test makes use of the milk-ejecting properties of
oxytocin.
• A rabbit in the first or second week of lactation is
anesthetized with urethane and pentobarbital. Usually,
artificial respiration is not necessary.
• One jugular vein is cannulated for injections. One of the six
ducts in a nipple of the rabbit is cannulated with a
hypodermic needle and connected with a Statham strain
gauge transducer.
• Two doses of the standard and the test preparation are
injected according to a randomized block or to a Latin
square design.
• Milk ejection pressure is recorded.