Saturday, 12 May 2012

Bioassay of acetylcholine

Bioassay of acetylcholine
Principle: Potency of the test sample is compared with that of the standard preparation.
There are several biological methods for its assay.

1. Rectus Abdominis Muscle of Frog: Dissect the rectus muscle and arrange the
assembly as per assay of d-tubocurarine. Plot log dose-response curve and find out the
potency of the sample of acetylcholine.


2. Cat’s Blood Pressure: A cat is anaesthetized with  suitable anaesthetic. The carotid
artery is cannulated for recording BP. Femoral vein is cannulated for injecting
acetylcholine. Trachea is cannulated for giving artificial respiration. Acetylcholine
produces a fall in BP by dilating peripheral blood vessels. This principle is utilised for its
bioassay. The extent to which BP falls due to the test sample is compared with the fall by
the standard preparation.

3. Guinea-pig Ileum: Guinea–pig is killed by a blow on the head and bled to death. The
abdominal wall is dissected out so as to isolate the ileum, the faecal matter, mesentery
and bood vessels are removed from the piece of ileum. It is ligated on both sides and
suspended in mammalian organ bath containing Tyrode solution maintained at 37.0oC
and oxygenated continuously. Acetylcholine contracts the ileum. This principle is utilised
for its bioassay. The extent of contraction produced by the test sample is compared with
the standard preparation of acetylcholine.

4. Anaesthetised Rats: Compare the extent of fall in BP of the test sample with that
produced by the standard preparation.

5.  Leech Muscle: Compare the contractions produced by the standard and test samples
on eserinised dorsal muscle of the leech. This muscle is highly sensitive (picograms) to
acetylcholine.

6. Isolate Heart Preparations: Rabbit’s auricle, frog’s heart, rabbit’s heart or venous
merceneria’s heart is used. Ach decreases the force and rate of the heart.

7. Rabbit’s Intestine and Tracheal Chain: Ach contracts these tissues.

(ii). Bioassay of adrenaline
Principle: Potency of the test sample is estimated by comparing the rise in blood pressure
of the test sample with the produced by the standard preparation of adrenaline.

Standard Preparation: It should fulfill all the tests of purity as per adrenaline monograph
in the Indian Pharmacopoeia. The specific rotation of 4% w/v solution of the standard
adrenaline in 0.1 N HCl should lie between –5 oC to 5  oC dilutions of adrenaline are
prepared by diluting it with saline. A suitable concentration is  1: 10,000. Test sample
solution is also prepared in saline.

1. Method using Anaesthetised Dog: A medium size dog (10-13 kg.) is anaesthetised
with pentobarbitone sodium in a dose of  25 mg/kg, i.v. Artificial  respiration is given
through tracheal tube, if necessary. BP is recorded on a kymograph after cannulating
carotid artery with arterial cannula and connecting the same with a mercury manometer.

The animal should be given 0.001-0.002 mg. of atropine sulphate to paralyse the vagi.
This paralysis is confirmed by stimulating vagi by electrical stimulation. There will be no
fall in BP in atropinised animal after electrical stimulation.

Adrenaline is injected into the femoral vein through venous cannula. Two successive
responses of the same dose of adrenaline are noted. The similar responses after the same
doses of adrenaline, is an indication that there is no variation in B.P., and that the animal
is now ready for the assay. Then the test and the standard samples are given in alternate
till both produce similar rise in BP. Injections are given at intervals of five minutes.
Potency of the test sample is compared with that of the standard sample.

2. On Spinal Cat: Refer pressor activity of posterior pitutary extract and apply the same
method.

3. Method Using Rabbit’s Duodenum: A rabbit weighing 2-3 kg. is killed by headblow
method and bled to death. The animal is dissected out, the abdominal organs are exposed
to isolate the duodenum. The duodenum is suspended in the inner bath of the mammalian
organ bath.

Inner bath is filled with Tyrode solution which is maintained at 37.5
oC. The muscle is
allowed to stabilize for 30 min. Rhythmic contractions are recorded by means of the
isotonic frontal writing lever on the kymograph. Adrenaline 
relaxes the duodenum and the same property is taken upto consideration for finding out
the potency of the test sample. Adrenaline is bioassayed by graphical method as shown in
Fig. 5.

4. De Jalon’s Method on Rat’s Uterus:  This method was first demonstrated by De
Jalon. A virgin female rat, weighing about 100-150 g. is used for the experiment. The rat
is killed by the headblow method and bled to death. The abdomen is opened and uterine
horns are isolated and placed in a petri dish containing de Jalon solution. Uterine horns
are ligated and suspended in a mammalian  organ bath containing modified Ringer
solution having the following composition is used: 

NaCl 9.0 gm; CaCl2 0.06 gm; KCl. 0.45 gm; Dextrose 0.5 gm NaHCO3 0.5 gm. and
distilled water 1000 ml. This solution abolishes the rhythmic contraction of the uterus. It
is maintained at 37oC and bubbled continuously with air.

Two identical contractions for 30 sec. with carbachol are recorded (0.75 mcg/ml). Then
selecting three different doses of standard  adrenaline and one intermediate dose test
adrenaline the percentage reduction of carbachol induced contractions are recorded.

5. Straub’s Method:  In this method, a glass cannula is passed through the aorta and
valves into the ventricle of heart so that the solution is pumped up and down in the
cannula with every beat. A graph paper attached behind the cannula to give an estimale of
the force of contraction of systole of the heart at every beat. The beat is recorded by
attaching the tip of the ventricle through a thread to a lever. This preparation has been
widely use for the detection of small quantity of adrenaline. Adrenaline increases the
force of contraction of the ventricle. The increased force produced by the test sample is
compared with that of the standard preparation

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