Saturday, 12 May 2012

Biological Assay of Oxytocin

Biological Assay of Oxytocin

Principle: The potency of oxytocin is determined by comparing its activity with that of
the Standard Preparation of oxytocin under the conditions of a suitable method of assay.
Standard Preparation: The Standard Preparation is the 4th International Standard for
Oxytocin, established in 1978, consisting of freeze-dried synthetic oxytocin peptide with
human albumin and citric acid (supplied in ampoules containing 12.5 Units), or another
suitable preparation the potency of which has been determined in relation to the

International Standard.

Suggested methods

Method A: By depression of the blood pressure in chicken — Anaesthetise a young
healthy adult cockerel weighing 1.2 to 2.3 kg with an anaesthetic  that will maintain a
  18prolonged and constant high blood pressure. Expose the gluteus primus muscle in one
thigh and cut and retract it to reveal the popliteal artery and crural vein. Cannulate the
popliteal artery and record the blood pressure on a suitable recorder calibrated for use
over a linear range. Cannulate the crural or brachial vein. Immediately before use prepare
a solution of the Standard Preparation in saline solution so that the volume to be injected
is between 0.1 ml and 0.5 ml. Record the blood pressure responses to the injection into
the cannulated vein of two doses of this solution; the doses should be such as to produce
clearly discriminated, precipitous, submaximal decreases in blood pressure; the required
doses normally lie between 20 and 100 milliUnits. The interval between injections should
be constant and lie between 3 and 10 minutes depending on the rate at which the blood
pressure returns to normal. Immediately before use dilute the preparation being examined
with saline solution so as to obtain responses similar to those obtained with the Standard
Preparation. The ratio between the two doses of the preparation being examined should
be the same as that between the two doses  of the Standard Preparation and this ratio
should be kept constant throughout the assay.
The two doses of the Standard Preparation and the two doses of the preparation being
examined should be given according to a randomised block or a Latin square design and
at least six responses to each should be recorded.
If the animal rapidly becomes insensitive to  the repeated injections of the solutions
another animal must be used. Measure all the responses and calculate the result of the
assay by standard statistical methods.
Method B: By contraction of the rat uterus — Inject 100 mcg of oestradiol benzoate
intramuscularly into a female rat weighing 120 to 200 g 18 to 24 hours before the assay.
Immediately before the assay  confirm by vaginal smear that  the rat is in oestrus or
preoestrus. Kill the rat and suspend one horn of the uterus in a bath containing a solution
of the following composition:
  Composition (% w/v)
Sodium chloride   0.662
Potassium chloride   0.045
Calcium chloride   0.007
Sodium bicarbonate   0.256
Disodium hydrogen phosphate   0.029
Sodium dihydrogen phosphate   0.003
Magnesium chloride   0.010
Dextrose  0.050
Maintain the bath at a temperature of 32o
 or at some other suitable temperature at which
spontaneous contractions of the uterus are  abolished and the preparation maintains its
sensitivity. Oxygenate the solution with a mixture of 95% of oxygen and 5% of carbon
dioxide and record the contractions of the muscle using a  suitable instrument giving a
linear response (for example an isotonic lever with a load not exceeding 2 g). Record the
  19contractions produced by the  addition to the bath of two doses of the Standard
Preparation suitably diluted with the above solution. The doses should be such as to
produce clearly discriminated, submaximal contractions; the required doses normally lie
between 10 and 50 micro Units per ml of  bath liquid. When maximal contraction has
been reached, replace the bath liquid by a fresh solution. The doses should be added at
regular intervals of 3 to 5 minutes depending upon the rate  of recovery of the muscle.
Dilute the preparation being examined so as to obtain responses on the addition of two
doses similar to those obtained with the Standard Preparation. The ratio between the two
doses of the preparation being examined should be the same as  that between the two
doses of the Standard Preparation and this  ratio should be kept constant throughout the
assay.
The two doses of Standard Preparation and the two doses of the preparation being
examined should be given according to a randomized block or a Latin square design and
at least six responses to each should be recorded.
Measure all the responses and calculate the result of the assay by standard statistical
methods.
Method C: By measurement of milk-ejection pressure in a lactating rat — Select a
lactating rat, in the third to twenty-first day after parturition and weighing about 300 g,
separate it from the litter and 30 to 60 minutes later anaesthetise  (for example, by the
intraperitoneal injection of a solution of  Pentobarbitone Sodium). Tie the rat to an
operating table, maintained at 37o
, by its hind legs leaving the front legs free. Cannulate
the trachea with a short polyethylene tube of internal diameter about 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 polyethylene tube of internal
diameter about 0.4 mm which is filled with saline solution and 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 polyethylene tube  of internal diameter
about 0.3 mm and external diameter about 0.6 mm, to a  depth sufficient to obtain
appropriate measurement of pressure (3 to 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
blood pressure in the rat) and fill the whole system with a 3.8% w/v solution of sodium
citrate or saline solution containing 50 Units of heparin sodium per ml to prevent clotting
of milk. After cannulation, inject a small volume (0.05 to 0.2 ml) of this solution into the
teat duct through the transducer to clear the milk from  the 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 about 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 solution.
  20Prepare a solution of the Standard Preparation and a solution of the preparation being
examined in saline solution so that the volume to be injected is between 0.1 ml and 0.4
ml. Choose two doses of the Standard Preparation such that the increase in milk-ejection
pressure is about 1.35 kPa for the lower dose and about 2.7 kPa for the higher 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 preparation being
examined with the same inter-dose ratio,  matching the effects of the doses of the
Standard Preparation as closely as possible. Inject the four doses (two doses of the
Standard Preparation and two doses of the preparation being examined) at intervals of 3
to 5 minutes. The two doses of Standard Preparation and the two doses of the preparation
being examined should be given according to a randomised block or a Latin square
design and at least four responses to each should be recorded. Measure all the responses
and calculate the result of the assay by standard statistical methods.

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