Saturday 12 May 2012

Bioassay of D-Tubocurarine

Bioassay of D-Tubocurarine

1. Rabbit Head-drop Method : Principle: d-Tubocararine hydrochloride is injected into
the marginal vein of a rabbit’s ear till the rabbit’s neck muscles are relaxed such that the
animal cannot hold its head up. The total amount of test sample required to produce the
endpoint is compared with the total amount of the standard sample required to produce
similar endpoint.


Selection of Rabbits:  Rabbits weighing 2 kg. are used. Animals should be free from
disease, obtained from a healthy colony and should be accustomed with the experimental
procedure.

Experimental Procedure:  Each rabbit is placed in a holder with its head protruding
outside. The head should be freely movable. Minimum 8 rabbits are used. They are
divided into two groups each containing 4 rabbits. First group will receive standard
sample and the second group will receive the sample under test. d-Tubocurarine solution
is injected at a constant speed by infusion apparatus through the marginal vein.


                                           (i)                               (ii)
Fig. 6: Rabbit head drop method for the bioassay of d-tubocurarine.
(i) : i.v. inj. of d-tubocurarine. (ii) : Head drop after injection.
Injection should be given at a rate of 0.4 ml/min and should take about 10 min. Infusion
is continued till the rabbit will not be in a position to hold its head erect or there will be
no response by focussing light on the eyes and the neck gets elongated and toneless.

Suitable dose of d-tubocurarine is 0.012% w/v in saline. Rabbits recover immediately
from the effect of curarization. During the expt.  there is a possibility or respiratory
embarrassment which is treated by injecting neostigmine methyl sulphate (0.05 mg.) and
atropine sulphate immediately through the marginal ear vein.

Cross-over test is carried out to minimise biological error due to animal variation. Those
rabbits which received the standard sample on the first day will be given test sample on
the second day of expt. and vice versa. Mean dose which produces head drop of the test
sample is compared with the mean dose of standard preparation.

2. Frog’s Rectus Abdominis muscle Preparation: A frog is pithed and laid on its back
on a cork covered board to which it is pinned. The skin covering the abdomen is cut away
and the rectus abdominis muscle of one side  is dissected from the pelvic girdle to its
insertion in the cartilage of the pectoral girdle. The muscle is then pinned to the cork by
four pins to keep its normal length while a  thread is sewn through each end. It is then
mounted in the organ bath containing frog’s Ringer solution which contains : NaCl, 6.5
gm.; KCl, 0.29 gm.; CaCl2, 0.24 gm.; NaHCO3, 0.4 gm.; glucose, 1.5 gm. and distilled
water 2000 ml. Oxygenation is  carried out to keep the tissue alive. The muscle is
stabilized for 30-45 min. in order to get critical quantitative response. The responses are
recorded using isotonic frontal writing lever with 1 G. tension.

Two similar contractions with the same concentration of acetylcholine are obtained.
Three doses of the standard sample and one  intermediate dose of the test sample are
selected and the reduction in height of  contraction induced by acetylcholine is noted
down.

Acetylcholine contraction is recorded on slow moving drum for 90 sec. d-Tubocurarine is
allowed to act for 30 sec. The percentage reduction at each dose levels is calculated and
log dose response curve of the standard drug is plotted. A linear response will be
obtained. The potency of test sample is calculated from the standard curve.
 

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