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Bioassay ofCorticosteroids ,

Insulin &Analgesics

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Biological assays of corticosteroids

Assay depending

on mineralocorticosteroid

activity

Assays

depending onglucocorticoid

activity

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Assay depending on mineralo-

corticosteroid activityurinary electrolyte balance in rats

Principle: Aldosterone makes sodium andwater retention and potassium loss leading

to decreased sodium and increasedpotassium in urine.

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urinary electrolyte balance in rats

Rats areadrenalectomized toremove endogenous

source of hormones

After treatment with adrenocortical

hormones, rats are put inmetabolic cages to collect urine

through a funnel.

Urine is analyzedfor Na+

and K+ ratio.

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Assays depending on glucocorticoid

activity

Liver glycogendeposition in rodents

Antiinflammatory

effectsGranuloma test in rats

Eosinopenia test in mice

Rat-paw edema

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Liver glycogen deposition in rodentsGlucocorticoids increase carbohydratemetabolism leading to rise in blood sugarlevel followed by its deposition in theliver as glycogen in a way proportional tothe dose of glucocorticoids.

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Antiinflammatory effectsGranuloma test in ratscotton pellets are implanted in the

axillae to produce granuloma. Thepellets will be considered as foreignbodies leading to an inflammatoryreaction. After 1 week, the cottonpellets become surrounded byfibrous tissues (granuloma), theyare isolated from the animal and

weighed (pellet + granuloma). Theanimal is given the glucocorticoidpreparation and the procedure isrepeated then the granuloma

weight is decreased

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Antiinflammatory effects

Eosinopenia test in mice:glucocorticoids reduce thenumber of eosinophils in aproportional way to the dose

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Antiinflammatory effectsRat -paw edema

(carrageenan-induced edema in the rathind paw)

Carrageenan is inflammatory materials usedto induce experimental inflammation due to

the release of inflammatory mediators(histamine, kinins and prostaglandins).

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Antiinflammatory effects

Carrageenan sodium gel (5 micro ml of1% solution) is injected S.C. in the

rat hind paw causing inflammation andedema after 1 hour.

The volume of edema in the hind pawis measured by volume displacement

using mercury plethysmography. Thehind paw is placed in mercury, causingmercury displacement. The displacedvolume is measured by mercury

manometer.

Rat-paw edema

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Antiinflammatory effects

Rat-paw edemaAnother group of rats is injectedI.P. by the anti-inflammatorydrug then, after 1 hour,carrageenan are injected into thehind paw. Anti-inflammatory drugs

reduce the volume of edema anddisplaced mercury.

Carrageenan

S.C

After 1 hour

Carrageenan

S.C

Test drug

I.P

+

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Antiinflammatory effectsRat-paw edema

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BIOLOGICAL ASSAY OF INSULIN

Mouse convulsionmethod

Rabbit bloodsugar method

The potency of insulin injection (expressed inInternational Units/ml) is determined by

comparing its hypoglycemic activity with thatproduced by the standard preparation of insulinusing one of the following methods

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Mouse convulsion method

Principle:This method is

based on thedetermination of thedose of the test

preparation, whichproduces hypoglycemicconvulsions in mice as

that produced bya dose of the standard insulinpreparation

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Mouse convulsion method

The standard insulin solution is diluted to a

concentration of 1 unit/ml, and the test solutionis also diluted to the same extent.

A 4-point assay is performed by injecting 2doses of the standard solution (S1 & S2) and 2doses of the test solution (T1 & T2) into fourgroups of mice.

Procedure

S1 S2 T1 T2

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Mouse convulsion method Mice are then kept at a constanttemperature (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 proportionalrelationship between %animals showingconvulsion and the potency of thepreparation.

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Rabbit blood sugar methodPrinciple: 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 fastedfor 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.

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Rabbit blood sugar method-Rabbits are randomly distributed into 4 groupsand deprived of food for not less than 18

hours before carrying out the assay.

Blood samples are withdrawn from themarginal ear vein and the average

concentration of blood glucose in mg/dl isdetermined in each group (initial blood sugar).

A 4-point assay is performed using the 4rabbit groups, where 2 doses of the test and 2doses of standard insulin preparation areinjected so that the dose of insulin does notexceed 0.5 unit/kg body weight.

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Rabbit blood sugar method

Following insulin injection, blood samples arewithdrawn from the marginal ear vein everyhour for 5 consecutive hours and the averageconcentration of blood glucose in mg/dl isdetermined in each group (final blood sugar).

For each dose of the test and standard insulin,the reduction in blood sugar level is calculatedand the potency of the test insulin is determined.

S1 S2 T1 T2

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Bioassay of Analgesics

Narcotic analgesicse.g. morphine,

methadone

Non-narcotic analgesicse.g. Salicylates,

diclofenac

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Bioassay of Narcotic analgesics

Mechanicalmethods

Thermalmethods

Tail clip method

Tail compressionmethod

Hot plate method

Tail flick method

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Tail clip methodPrinciple: The mouse tail (as well as the ear) isnot covered by hair so it is very sensitive topain due to the presence of sensory nerves inthe skin.

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

Test A group of mice is treated withthe 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

Control

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Tail compression methodPrinciple: This test uses an instrument(analgesiometer) producing a gradual increasingpressure on the tail which can be measured.

Procedure:*The pressure is gradually increasedtill a certain threshold pressure (e.g.20 mm Hg) is reached at which the

animal squeaks.*The mean threshold pressure incontrol animals is determined.*The drug under test is injected I.P.

and the mean threshold pressure isdetermined after 30, 60 120 and180 minutes. If the thresholdpressure increases by 2 folds (e.g.

40 mm Hg) then the drug may be ananalgesic.

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Hot plate method

*Principle:*Principle:-Mouse on 50C hot plate

licks, stands on its feet

-Inject analgesic IP then put on hot plate again

mouse takes longer time to beginlicking and standing

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Hot plate method

Procedure*Mouse is put in a glass cylinder kepton a hot plate at 55Cand the reaction

time is calculated (time frominsertion of the animal inside thecylinder until it licks its feet ortries to jump out of the cylinder).

* The mean reaction time in a controlgroup is usually < 1 minute.* The drug under test (X) is injectedinto another group of mice and the

reaction time is determined after 10,30, 60, 90, 120 and 180 minutes. Ifthe reaction time is increased by 3folds (3 minutes for e.g.), the drug

is considered as an analgesic

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Tail flick methodElectricshock

*The rats tail is used, and an electriccurrent is allowed to pass through a coiluntil it becomes bright red.

*The reaction time, which is the timeelapsed between closing of electriccircuit and withdrawal of tail, is

recorded.

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Bioassay of Non Narcotic analgesics

Writhing method

In this method, moderate pain is induced in miceby 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 IPirritationstretching & writhing (twist due

to pain) count no. of writhings-Give Aspirin then glacial acetic acid count no. of writhings

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Writhing method

3.) Procedure:3.) Procedure:1. Weigh mouse2. Inject mouse IP with 1ml/100 g of

10% glacial acetic acid

3. Record the onset and the count ofwrithings4. Give Aspirin orally to another mouse

in a dose of 50 mg/kg

5. After 30 min inject glacial aceticacid IP

6. Record the onset and count ofwrithings and compare

If no writhing occurs, the drug maybe an analgesic.

It is a quantal assay.

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