Estimation of unknown concentration of test drug

1. Assay of
Histamine
Presenter:
Dr Arun Singh
Senior Resident
Department of Pharmacology
SMS Medical College, Jaipur

2. Aim: To determine unknown concentration of
histamine by using guinea pig ileum.

3. Background
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.
The assay of the preparation is based on the magnus method (1904).

4. Guinea pig ileum has 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 × 10-6to 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.

5. 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 Mwt. on the reagent bottle may vary with different salt compositions)

6. Precautions
before
Experimentation
Add Add the calcium chloride at the end of PSS preparation (to avoid
any precipitation: PSS should be clear)
Prepare Prepare PSS for the experiment, while taking exact quantity of
chemicals (1% variability is acceptable)
Balance Balance the writing lever horizontally with the help of load
Clean Clean the organ bath before starting the experiment especially
inner organ bath (chances of presence of previous drug used)

7. Try to minimize the handling of tissue (especially at the middle
part)
Try
Always use the finger to hold the tissue instead of forceps
Use
Maintain the dose cycle properly (tissue sensitivity depends on
the cycle)
Maintain
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).
Avoid

8. Methods
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.

9. 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 (also see in identification and collection of tissue section).

10. 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.

11. 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 3-point, 4-point or
any other design mentioned. Then, make the DRC of standard
and test drug then plan for the selected experimental design
accordingly such as for 3-point select 2 response of standard
and 1 response of test whereas for 4-point select 2 responses
of standard and test each.

12. Step X: Prepare the complete graph and calculate for determining
the unknown concentration of given drug.
Note: Rat or rabbit ileum bioassay procedure is same as described
in the Guinea pig ileum. But, tissue shows variable spontaneous
response hence the tissue should be relaxed properly.

13. Calculation and Result
• The calculation and graph depend on the method/design of
experimentation adopted either 3- point, 4-point or matching, etc. (see the
example given in bioassay chapter for calculation).

14. Inference
• Write the findings of the study.

15. Discussion
• Ileum is preferred for the experiment because of less mesentery attached to ileum, and
nearly all receptors are present.
• But, 10 cm of ileum attached to the cecum contains more of α-excitatory receptor is
present which should be excluded.
• Intestine is supplied by both parasympathetic and sympathetic nerves.

16. • Muscarinic receptors are predominant in parasympathetic action whereas
inhibitory activity is mediated by the sympathetic α and β- receptors.
• It is reported that inhibitory α-receptors is present located presynaptically
whereas β-receptor (especially β1) are present on the smooth muscle fibers.
• Fasting leads to less possibility of fecal matter present at the site.

17. • The selection of tissue depends on the drug such as guinea pig ileum is most
sensitive for the histamine or related compounds.
• Always select the tissue which is most sensitive to the drug.

18. For example:
If the drug is ACh, then select the dorsal leech muscle (if
available) or frog rectus abdominis muscle, etc.
For the intestinal preparation most commonly used species
are guinea pig and rabbit.
Spontaneous activity of the tissue is reduced by performing
the experiment 5-7°C lower than the body temperature.

19. Thank You