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Study Material

The document outlines an experiment for the bioassay of histamine using guinea pig ileum through a matching method, emphasizing the sensitivity of the ileum to histamine and the role of H1 and H2 receptors. It details the materials, methods, and precautions necessary for conducting the experiment, including steps for tissue preparation and drug administration. The conclusion assesses whether the test sample meets quality control standards based on the response compared to a standard dose of histamine.

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6 views3 pages

Study Material

The document outlines an experiment for the bioassay of histamine using guinea pig ileum through a matching method, emphasizing the sensitivity of the ileum to histamine and the role of H1 and H2 receptors. It details the materials, methods, and precautions necessary for conducting the experiment, including steps for tissue preparation and drug administration. The conclusion assesses whether the test sample meets quality control standards based on the response compared to a standard dose of histamine.

Uploaded by

Bhavya
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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Experiment – VII

Bioassay of histamine using guinea pig ileum by matching method

Aim: In Silico demonstration of bioassay of histamine using guinea pig ileum by matching
method (using Ex-Pharm / Ex-Physio Series Software).

Principle:

Guinea pig ileum is most sensitive to histamine. Histamine, mainly act through the H1 and H2 receptor.
Contractile response of histamine to the ileum is caused by the H1 receptor found in the ileum, bronchi
and capillaries. Besides the triple response caused by it, histamine has spasmogenic response on
intestinal smooth muscle. The assay of the preparation is based on the magnus method (1904).
Guinea pig ileum has the spontaneous activity and its specificity is improved by using atropine or
mepyramine in tyrode. In the assay of histamine, atropine (2 × 10–6 to 10–7 M) and for acetylcholine
(ACh) assay mepyramine (5 × 10–6 to 2 × 10–7) is used for the enhancement of the specificity. Some
of the endogenous polypeptides like angiotensin, bradykinin and substance P are also sensitive to
guinea pig ileum.

Materials and Method:

Animal/tissue : Guinea Pig/ileum


PSS : Tyrode/Ringer
Lever : Frontal writing
Tension/load : upto 1 gm
Magnification : 7-10x
Aeration air : O2/Carbogen
Temperature : 32-37°C
Drug : Histamine (Mwt: 307.14) (Always check M wt. on the reagent bottle may vary with different salt
compositions)

Precautions before Experimentation:

 Clean the organ bath before starting the experiment specially inner organ bath (chances
 of presence of previous drug used).
 Balance the writing lever horizontally with the help of load.
 Prepare PSS for the experiment, while taking exact quantity of chemicals (1% variability is
 acceptable).
 Add the calcium chloride at the end of PSS preparation (to avoid any precipitation: PSS should be
clear)
 Try to minimize the handling of tissue (especially at the middle part).
 Always use the finger to hold the tissue instead of forceps.
 Maintain the dose cycle properly (tissue sensitivity depends on the cycle).
 Avoid contact of very high dose of the drug; tissue may loss sensitivity due to “tachyphylaxis”.
 (tissue may remain in contractile stage, in presence of high drug concentration).
Method:

Step I: Keep the animal (guinea pig) for fasting at least for 24-48 hr.

Step II: Sacrifice the guinea pig by stunning (a strong blow) on the head then, keep the animal on the
dissecting board (DB).

Step III: Fix guinea pig on the DB by tying its legs with the help of thread.

Step IV: Cut open the abdomen of guinea pig by a vertical midline incision after a small horizontal cut,
and then expose the abdominal contents.

Step V: Identification of the ileum is done by two ways:

(1) Identify the cecum then come back at least of 10 cm,


or
(2) Identify stomach, then go forward to identify the ileum 10 cm before cecum.

Step VI: Take out the 2 -3 cm or as desired length of the ileum and remaining should be kept in
refrigerator for the future use.

Step VII: The ileum is trimmed away from mesentery or attached tissues, then clean any waste contains
of the ileum by a gentle push of PSS by the help of syringe (preferably PSS warmed at 30-35°C.

Step VIII: Then, the ileum is attached to the one end with the hook attached to the tissue holding arm
and the other end is tie to the lever. (Attachment of the tissue should be in the direction of the
intestine in vivo as far as possible).

Step IX: The experimental design is selected matching method. Then, make the DRC of standard and test
drug then plan for the selected experimental design accordingly such as for matching where one dose is
selected from the DRC which come under 25-75% response. Then take a response of the test solution.
Match the response with selected dose of the standard either by increasing or decreasing the dose of
the test till the response matches with the selected dose of the standard.

Step X: Prepare the complete graph and calculate for determining the unknown concentration of given
drug using the formula:

Dose of standard

Relative potency = -----------------------

Dose of Test
Result:

Histamine Dose Standard Volume (ml) Response (mm)

Dose
Response
Curve of
standard

Standard
Test
Standard
Test
Standard
Test
Standard
Test

Calculation:

Dose of standard

Relative potency = -----------------------

Dose of Test

Conclusion: The test sample does/ does not pass the quality control as the standard range is (X – Y).

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