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| LETTER TO THE EDITOR |
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| Year : 2013 | Volume
: 45
| Issue : 3 | Page : 312-313 |
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Isolated chick ileum for bioassay of acetylcholine
Parthasarathy Nirmala, Kannapiran Elandevan, Natesan Chidambaram, Asirvatham Sylvia Santhakumari
Department of Pharmacology, Rajah Muthiah Medical College, Annamalai University, Chidambaram, Tamil Nadu, India
| Date of Web Publication | 15-May-2013 |
Correspondence Address:
Parthasarathy Nirmala
Department of Pharmacology, Rajah Muthiah Medical College, Annamalai University, Chidambaram, Tamil Nadu
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/0253-7613.111927
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 |
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.
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 | |  |
| 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. 
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| 2. | Bolton TB. Intramural nerves in the ventricular myocardium of the domestic fowl and other animals. Br J Pharmacol Chemother 1967;31:253-68.
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| 3. | Darroch S, Irving HR, Mitchelson FJ. Characterisation of muscarinic receptor subtypes in avian smooth muscle. Eur J Pharmacol 2000;402:161-9.
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| 4. | Chand N, Eyre P. The pharmacology of anaphylaxis in the chicken intestine. Br J Pharmacol 1976;57:399-408.
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[Figure 1]
[Table 1]
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