histamine bioassay

1. BIOASSAY OF HISTAMINE BIOASSAY OF HISTAMINE

GIRIJA MAGANTI
MPHARM (PHARMACOLOGY)

2. What is histamine?What is histamine?
•Histamine is a small ,water soluble
molecule or amine autocoid or locally
acting hormone which mediates its effects
by binding to receptors H1,H2,H3,H4.

3. Histamine synthesisHistamine synthesis

4. Site of synthesis and storage:Site of synthesis and storage:
Histamine is synthesized and stored in the
following sites:
1- Neurons in the brain
2- Entero chromaffin cells in the gastric
mucosa
3- Mast cells

5. Non mast cell sources of histamine Non mast cell sources of histamine
in the body in the body
1- Brain: (functions as neurotransmitter)
2- Entero chromaffin cells (EC) in the
stomach
(function: stimulates HCL secretion by
parietal cells of the stomach)

6. RELEASE OF HISTAMINE FROM RELEASE OF HISTAMINE FROM
MAST CELLMAST CELL
1.Immune mediated 1.Immune mediated

7. 2.Non immune mediated 2.Non immune mediated
•Certain drugs such as morphine and
tubocurarine, can displace histamine from
mast cells. This type of release does not
require prior exposure.

8. Pharmacological actions of Pharmacological actions of
histaminehistamine
•The pharmacological actions of histamine
depend on the tissue and type of receptor
present at the area of release

9. Histamine receptorsHistamine receptors
R subtypeDistribution Action Agonist Antagonist
H1 Sm. MuscleContraction of
GIT,bronchoco
nstricton.
Hist. Mepyramine
Cyproheptadine
H2 Gastric
mucosa
Acid release Hist.Cimetidine
Ranitidine
H3 Presynaptic Autoregulation
of histamine
release
Hist.Thioperamide
H4 Eosinophils
Neutrophils
CD4T cells
Modulate the
production of
blood cells &
cytokines
Hist. Thioperamide

10. Symptoms associated with histamine Symptoms associated with histamine
release from mast cellsrelease from mast cells
•Mild cutaneous release
•Moderate release
•Severe release (anaphylactic)

•erythema, urticaria, and/or itching
•skin reactions, tachycardia, moderate
hypotension, mild respiratory distress
•severe hypotension, ventricular
fibrillations, cardiac arrest, bronchospasm,
respiratory arrest

11. DefinitionDefinition
•Estimation of the conc / potency of a
substance by measuring its biological
response in living systems
•i.e.Observation of pharmacological effects
on
•[1] living tissues, or cells
•[2] microorganisms
•[3] animals

12. Bioassay of histamine can be done by
recording
1. Contractions of isolated guinea pig ileum.
2. BP fall in anaesthetised cat or dog .

13. Bioassay using guinea pig ileumBioassay using guinea pig ileum
•Bioassay of histamine on isolated guinea
pig ileum can be determined by
Matching bioassay
Interpolation bioassay
Bracketing assay
Multiple point assays

14. Ileum Ileum
•3/5 of intestine
•Empties in the large intestine via ileocecal
valve
•Bile salts, vitamin B12, water and
electrolytes absorption
•Doesn’t have myogenic contraction
•More sensitive to histamine action

15. Histamine receptors in ileum Histamine receptors in ileum
•H1 receptors
•Receptor type: G-protein-coupled receptor
•Agonist: Histamine
•MOA: ++ G-protein → ++phspholipase C → splitting
of PIP2 into 1) DAG that increases the opening of
calcium channels 2)IP3 which increases calcium
mobilization from sarcoplasmic stores;
DAG & IP3 lead to increase in the intracellular
concentration of calcium and smooth muscles
contraction.

16. REQUIREMENTSREQUIREMENTS
•Instruments:thermo statically controlled
organbath,chymograph,aerator
•Physiological solution:tyrode’s solution
•Temperature:32°c
•Animal :guinea pig
•Standard histamine solution(10µg/ml)

17. Preparing standardPreparing standard
•Take 10mg of histame + 10ml of water
(1000µg/ml)
•Take0.1ml and dilute with
10mlwater(10µg/ml)

19. THREE POINT BIOASSAYTHREE POINT BIOASSAY
•2STANDARD
•1 TEST

20. BIOASSAY USING BIOASSAY USING
ANAESTHETISED CAT OR ANAESTHETISED CAT OR
DOGDOG
•Cat or dog is anaesthetised with chloralose
or barbiturate and prepared for recording of
BP
•Sensitivity is determined by injecting
standard solution of 0.05,0.1,0.15µg of
histamine base per kg bodyweight is given
for 5min interval

21. •A fixed dose of standard producing a fall in
BP about 20mmHg is injected with
changing doses of test at regular intervals
and matching assay is done

22. MECHANISM OF BP FALLMECHANISM OF BP FALL
• histamine binds wth histamineH
1
receptor of
endothelium causes release of EDRF
(endothelium derived relaxing factor). This EDRF
diffuses out and reaches the smooth muscle of the
arteriole and causes generation of cyclic-GMP →
causing reduction of Ca
++
in smooth muscle
relaxation of the smooth muscle→ arteriolar
dilatation. Another possibility is that combination
of H
1
with the Histamine causes release of
PGI
2
which causes vasodilatation.