Bioassay of Corticosteroids , Insulin & Analgesics

Biological assays of corticosteroids

Assay depending on mineralo corticosteroid activity

Assays depending on glucocorticoid activity

Assay depending on mineralocorticosteroid activity

urinary electrolyte balance in rats Principle: Aldosterone makes sodium and water retention and potassium loss leading to decreased sodium and increased potassium in urine.

urinary electrolyte balance in rats

Rats are adrenalectomized to remove endogenous source of hormones

After treatment with adrenocortical hormones, rats are put in metabolic cages to collect urine through a funnel.

Urine is analyzed for Na+ and K+ ratio.

Assays depending on glucocorticoid activity

Liver glycogen deposition in rodents

Antiinflammatory effects
Granuloma test in rats Eosinopenia test in mice Rat-paw edema

Liver glycogen deposition in rodents

Glucocorticoids increase carbohydrate metabolism leading to rise in blood sugar level followed by its deposition in the liver as glycogen in a way proportional to the dose of glucocorticoids.

Antiinflammatory effects
cotton pellets are implanted in the axillae to produce granuloma. The pellets will be considered as foreign bodies leading to an inflammatory reaction. After 1 week, the cotton pellets become surrounded by fibrous tissues (granuloma), they are isolated from the animal and weighed (pellet + granuloma). The animal is given the glucocorticoid preparation and the procedure is repeated then the granuloma weight is decreased

Granuloma test in rats

Antiinflammatory effects
Eosinopenia test in mice:
glucocorticoids reduce the number of eosinophils in a proportional way to the dose

Antiinflammatory effects
Rat-paw edema
(carrageenan-induced edema in the rat hind paw) Carrageenan is inflammatory materials used to induce experimental inflammation due to the release of inflammatory mediators (histamine, kinins and prostaglandins).

Antiinflammatory effects
Rat-paw edema
Carrageenan sodium gel (5 micro ml of 1% solution) is injected S.C. in the rat hind paw causing inflammation and edema after 1 hour. The volume of edema in the hind paw is measured by volume displacement using mercury plethysmography. The hind paw is placed in mercury, causing mercury displacement. The displaced volume is measured by mercury manometer.

Antiinflammatory effects
Rat-paw edema
Another group of rats is injected I.P. by the anti-inflammatory

drug then, after 1 hour, carrageenan are injected into the hind paw. Anti-inflammatory drugs reduce the volume of edema and displaced mercury.
Test drug I.P Carrageenan S.C

After 1 hour Carrageenan S.C

Antiinflammatory effects
Rat-paw edema

BIOLOGICAL ASSAY OF INSULIN

The potency of insulin injection (expressed in International Units/ml) is determined by comparing its hypoglycemic activity with that produced by the standard preparation of insulin using one of the following methods

Mouse convulsion method

Rabbit blood sugar method

Mouse convulsion method

Principle: This method is
based on the determination of the dose of the test preparation, which produces hypoglycemic convulsions in mice as that produced by a dose of the standard insulin preparation

Mouse convulsion method

Procedure
The standard insulin solution is diluted to a concentration of 1 unit/ml, and the test solution is also diluted to the same extent. A 4-point assay is performed by injecting 2 doses of the standard solution (S1 & S2) and 2 doses of the test solution (T1 & T2) into four groups of mice.

S1

S2

T1

T2

Mouse convulsion method

Mice are then kept at a constant temperature (29-35C) and observed for 1.5 hours following insulin injection. The number of animals showing convulsion is recorded. The potency of the test preparation is determined by a specific statistical method based on the direct proportional relationship between %animals showing convulsion and the potency of the preparation.

Rabbit blood sugar method

Principle: insulin lowers blood sugar level,
and the degree of blood sugar lowering is proportional to the potency of the preparation.

Procedure
12 to 20 healthy rabbits (1.5 to 2 kg) are used. A preliminary test is carried out to exclude any rabbit liable to convulsion, where rabbits are fasted for 18 hours then each rabbit is injected s.c. with one unit of insulin and observed for 5 hours; any rabbit showing convulsions is excluded.

Rabbit blood sugar method

-Rabbits

are randomly distributed into 4 groups and deprived of food for not less than 18 hours before carrying out the assay.

Blood samples are withdrawn from the marginal ear vein and the average concentration of blood glucose in mg/dl is determined in each group (initial blood sugar). A 4-point assay is performed using the 4 rabbit groups, where 2 doses of the test and 2 doses of standard insulin preparation are injected so that the dose of insulin does not exceed 0.5 unit/kg body weight.

Rabbit blood sugar method

S1

S2

T1

T2

Following insulin injection, blood samples are withdrawn from the marginal ear vein every hour for 5 consecutive hours and the average concentration of blood glucose in mg/dl is determined in each group (final blood sugar). For each dose of the test and standard insulin, the reduction in blood sugar level is calculated and the potency of the test insulin is determined.

Bioassay of Analgesics

Narcotic analgesics e.g. morphine, methadone

Non-narcotic analgesics e.g. Salicylates, diclofenac

Bioassay of Narcotic analgesics

Mechanical methods

Thermal methods

Tail clip method Tail compression method

Hot plate method Tail flick method

Principle: The mouse tail (as well as the ear) is not covered by hair so it is very sensitive to pain due to the presence of sensory nerves in the skin.
Control

Tail clip method

An artery clip (clamp) is applied to the base of the mouse tail for 30 seconds. The animal will feel pain and will try to get rid of the clip A group of mice is treated with the test drug, and the clip is applied to the tail after 30, 60,120 and 180 minutes If the animals response to the clip is abolished or delayed,then the drug may have analgesic properties. The ED50 is calculated

Test

Tail compression method

Principle: This test uses an instrument (analgesiometer) producing a gradual increasing pressure on the tail which can be measured.
Procedure: *The pressure is gradually increased till a certain threshold pressure (e.g. 20 mm Hg) is reached at which the animal squeaks. *The mean threshold pressure in control animals is determined. *The drug under test is injected I.P. and the mean threshold pressure is determined after 30, 60 120 and 180 minutes. If the threshold pressure increases by 2 folds (e.g. 40 mm Hg) then the drug may be an analgesic.

Hot plate method

-Mouse on 50C hot plate licks, stands on its feet -Inject analgesic IP then put on hot plate again mouse takes longer time to begin licking and standing

*Principle:

Hot plate method

Procedure
*Mouse is put in a glass cylinder kept on a hot plate at 55Cand the reaction time is calculated (time from insertion of the animal inside the cylinder until it licks its feet or tries to jump out of the cylinder). * The mean reaction time in a control group is usually < 1 minute. * The drug under test (X) is injected into another group of mice and the reaction time is determined after 10, 30, 60, 90, 120 and 180 minutes. If the reaction time is increased by 3 folds (3 minutes for e.g.), the drug is considered as an analgesic

Electric shock

Tail flick method

*The rats tail is used, and an electric current is allowed to pass through a coil until it becomes bright red. *The reaction time, which is the time elapsed between closing of electric circuit and withdrawal of tail, is recorded.

Bioassay of Non Narcotic analgesics

Writhing method
In this method, moderate pain is induced in mice by chemicals like acetic acid (300 mg/kg) producing seizures of abdominal cramps (i.e. cramps in the abdominal skeletal muscles) after 15 min (positive writhing response).
-Give Glacial acetic acid IP

irritation stretching & writhing (twist due to pain) count no. of writhings -Give Aspirin then glacial acetic acid count no. of writhings

Writhing method
3.) Procedure: 1. Weigh mouse 2. Inject mouse IP with 1ml/100 g of 10% glacial acetic acid 3. Record the onset and the count of writhings 4. Give Aspirin orally to another mouse in a dose of 50 mg/kg 5. After 30 min inject glacial acetic acid IP 6. Record the onset and count of writhings and compare

If no writhing occurs, the drug may be an analgesic. It is a quantal assay.