BIOASSAY OF HISTAMINE BIOASSAY OF HISTAMINE

GIRIJA MAGANTI

MPHARM (PHARMACOLOGY)

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.

Histamine synthesisHistamine synthesis

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

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)

RELEASE OF HISTAMINE FROM RELEASE OF HISTAMINE FROM MAST CELLMAST CELL1.Immune mediated 1.Immune mediated

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.

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

Histamine receptorsHistamine receptorsR subtype Distribution Action Agonist Antagonist

H1 Sm. Muscle Contraction of GIT,bronchoconstricton.

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

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

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

Bioassay of histamine can be done by recording

1. Contractions of isolated guinea pig ileum.

2. BP fall in anaesthetised cat or dog .

Bioassay using guinea pig ileumBioassay using guinea pig ileum

• Bioassay of histamine on isolated guinea pig ileum can be determined by

Matching bioassayInterpolation bioassayBracketing assayMultiple point assays

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

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.

REQUIREMENTSREQUIREMENTS

• Instruments:thermo statically controlled organbath,chymograph,aerator

• Physiological solution:tyrode’s solution

• Temperature:32°c

• Animal :guinea pig

• Standard histamine solution(10µg/ml)

Preparing standardPreparing standard

• Take 10mg of histame + 10ml of water

(1000µg/ml)

• Take0.1ml and dilute with 10mlwater(10µg/ml)

THREE POINT BIOASSAYTHREE POINT BIOASSAY

• 2STANDARD

• 1 TEST

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

• 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

MECHANISM OF BP FALLMECHANISM OF BP FALL

• histamine binds wth histamineH1 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 H1 with the Histamine causes release of

PGI2 which causes vasodilatation.