Isolated chick ileum for bioassay of acetylcholine

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, CaCl₂ 2.0 mM, MgCl₂ 0.5 mM, KH₂PO₄ 1.2 mM, NaHCO₃ 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

Three point bioassay with ACh was carried out in four experiments. Dose responses were recorded for Ach-two doses of standard (S₁ and S₂) and one dose of test (T) with an intermediate response between S₁ and S₂. Three sets of responses using S_1, S₂, 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, S₂, and T) were substituted in the following formula to determine the concentration of the Test solution.

s₁ x antilog {T-S₁/S₂-S₁ x log s₂/s₁} where s₁ = lower standard dose; s₂ = higher standard dose; t = test dose; S₁ = response of s_1; S₂ = response of s₂; T = response of test (t).

Results from a typical experiment using S₁ (2 μg) and S₂ (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

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 M₃ 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% O₂ + 5% CO₂) 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.