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LETTER TO THE EDITOR
Year : 2013  |  Volume : 45  |  Issue : 3  |  Page : 312-313
 

Isolated chick ileum for bioassay of acetylcholine


Department of Pharmacology, Rajah Muthiah Medical College, Annamalai University, Chidambaram, Tamil Nadu, India

Date of Web Publication15-May-2013

Correspondence Address:
Parthasarathy Nirmala
Department of Pharmacology, Rajah Muthiah Medical College, Annamalai University, Chidambaram, Tamil Nadu
India
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DOI: 10.4103/0253-7613.111927

PMID: 23833386

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How to cite this article:
Nirmala P, Elandevan K, Chidambaram N, Santhakumari AS. Isolated chick ileum for bioassay of acetylcholine. Indian J Pharmacol 2013;45:312-3

How to cite this URL:
Nirmala P, Elandevan K, Chidambaram N, Santhakumari AS. Isolated chick ileum for bioassay of acetylcholine. Indian J Pharmacol [serial online] 2013 [cited 2014 Nov 1];45:312-3. Available from: http://www.ijp-online.com/text.asp?2013/45/3/312/111927


Sir,

Current restrictions in experiments with laboratory animals prompted the search for alternate tissues for biological testing. Tissues from poultry, sheep, goats, cattle, fish, etc., usually consumed for food, were suggested. [1] We investigated ileum from chicks sacrificed for food as a possible alternative in 20 experiments.

The small intestine in chick is a long and uniform in the diameter. Its circular muscles are 3 times thicker than the longitudinal muscles.

From a local government registered meat shop fresh intestine of chicken was collected into a flask containing 500 ml "chick" solution, transported immediately to laboratory and kept aerated. Composition of chick solution: NaCl 118.4 mM, KCl 4.6 mM, CaCl 2 2.0 mM, MgCl 2 0.5 mM, KH 2 PO 4 1.2 mM, NaHCO 3 25 mM, glucose 11.1 mM, and sucrose 13.2 mM. [2]

In initial experiments with Tyrode (3) or Chick Solution (10) the bath was maintained at 37°C. When the tissue responses to acetylcholine (ACh) were not uniform, taking a long time for relaxation, we changed to chick solution maintained at 42°C [2] in the last 7 experiments.

The piece of ileum (approximately 2 cm) with contents washed with chick solution was mounted in 10 ml organ bath kept at 42°C and aerated. It was rested for 1 h under 1 g tension. The isotonic responses of the tissue were recorded on kymograph with frontal ink writing lever at a magnification of seven. ACh, atropine (ATR) and all other chemicals used were of analytical reagent (AR) grade.

At 1 μg doses the preparation responded to ACh, but not histamine and 5 hydroxy tryptamine (5-HT). ACh was used in all experiments applying a cycle of contact to ACh for 30 s at 3 min intervals. Ach (0.5-8 μg) produced dose dependent contractions added to 10 ml bath.

Competitive antagonism of ACh to ATR was shown in 3 experiments. [Figure 1] illustrates two responses to ACh (2 μg) and blockade by ATR (2 μg), added 30 s before, followed by the slow recovery.
Figure 1: Chick ileum (competitive antagonism of acetylcholine [ACh] by atropine [ATR]) physiological chick solution temperature: 42°C ACh: 2 μg ATR: 2 μg

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Three point bioassay with ACh was carried out in four experiments. Dose responses were recorded for Ach-two doses of standard (S 1 and S 2 ) and one dose of test (T) with an intermediate response between S 1 and S 2. Three sets of responses using S 1, S 2 , and T were recorded in randomized Latin square fashion to allow for the fluctuating sensitivity of the tissue. The mean heights of all the three responses (S 1, S 2 , and T) were substituted in the following formula to determine the concentration of the Test solution.

s 1 x antilog {T-S 1 /S 2 -S 1 x log s 2 /s 1 } where s 1 = lower standard dose; s 2 = higher standard dose; t = test dose; S 1 = response of s 1; S 2 = response of s 2 ; T = response of test (t).

Results from a typical experiment using S 1 (2 μg) and S 2 (4 μg), concentration of the test was determined with the above formula. In the experiment of [Table 1], the actual value of T was 400 mg and by bioassay, it was 353.5 mg. that is 88.4% of the actual strength.
Table 1: Three point bioassay of acetylcholine on isolated chick ileum


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From four such experiments, bioassay values were 80-100% of the actual values supporting its degree of accuracy and reproducibility

Biological testing is a cheap and specific prelude to identify the nature of receptors and quantifying the unknown substance. In chick ileum M 3 muscarinic receptors were reported. [3] Spiral ileal strips from fowl sensitized with albumin, contract many times more with ACh than with histamine and 5-HT. [4] Chick ileum maintains good stability with reproducibility. However, it needs time with a 1 h resting period before the assay and 2-3 min interval after each contraction. Use of carbogen (95% O 2 + 5% CO 2 ) for gassing [2] was not available with us. It may help in improving the response. Chick ileum can be used as an alternative for tissues from laboratory animals for bioassay and demonstrations.

 
  References Top

1.Haranath PS. 'Changing Face of Pharmacology' - Souvenir of 42 nd Annual Conference of Indian Pharmacological Society 10-12 December. Kolkata 2009. p. 43-4.  Back to cited text no. 1
    
2.Bolton TB. Intramural nerves in the ventricular myocardium of the domestic fowl and other animals. Br J Pharmacol Chemother 1967;31:253-68.   Back to cited text no. 2
    
3.Darroch S, Irving HR, Mitchelson FJ. Characterisation of muscarinic receptor subtypes in avian smooth muscle. Eur J Pharmacol 2000;402:161-9.  Back to cited text no. 3
    
4.Chand N, Eyre P. The pharmacology of anaphylaxis in the chicken intestine. Br J Pharmacol 1976;57:399-408.  Back to cited text no. 4
    


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