BIOASSAY OF INSULIN Notes Pharmacology

Bioassay-of-insulin

BIOASSAY OF INSULIN

Standard preparation and units : it is a pure, dry and crystalline insulin in state.

One unit contain 0.04082 milligram of insulin.

The unit is specified by the ministry of health, Government of India and is equivalent to the international unit.

Preparation of standard solution: Accurately weigh 20 units of insulin and dissolve it in Normal saline.

Acidify it with hydrochloric acid [HCL] to pH 205.

Add 0.5% phenol as preservative.

Now Add 1.4% to 1.8% of glycerine

The final volume should contain 20 units/ml.

Store the solution in a cool place and use it within six months of time period.

PREPARATION OF TEST SAMPLE SOLUTION:

The solution of test sample is prepared in the same way as the standard solution described below.

Accurately weigh 20 units of insulin and dissolve it in Normal saline.

Acidify it with hydrochloric acid [HCL] to pH 205.

Add 0.5% phenol as preservative.

Now Add 1.4% to 1.8% of glycerine

The final volume should contain 20 units/ml.

Store the solution in a cool place and use it within six months of time period.

BIOASSAY OF INSULIN THERE IS MAINLY TWO TYPES OF ANIMALS ARE USED:
The model is based on animals.

(1)  MOUSE METHOD

(2)  RABBIT METHOD

bioassay-of-insulin-mouse-method

PROCEDURE OF BIOASSAY OF INSULIN BY MOUSE METHOD

Mice shows characteristic convulsions after SC. injection of insulin at elevated temperature.

The percentage convulsions produced by the TEST and STANDARD preparation are compared.

EXPERIMENTAL PROCEDURE BIOASSAY OF INSULIN BY MOUSE METHOD:

Minimum 100 Mice are selected weighing between 18-22 grams.

The same species and same strain of mouse are used.

They should be maintained on a constant Diet.

They should be fasted Eighteen hours (18 Hrs.) prior to the experiment.

PREPARATION OF STANDARD AND SAMPLE DILUTIONS

 MOUSE METHOD  
25 Mice25 Mice25 Mice25 Mice
StandardStandardTest sampleTest sample
Dilution
(0.064 units/ml)
Dilution
(0.096 units/ml)
DilutionDilution

Dilutions are prepared with sterile saline solution, so as to contain 0.064 Units/ml [standard dilution I] and 0.096 Units/ml [standard dilution II].

mouse-method

TEST SAMPLE SOLUTION

Dilutions are prepared with sterile saline solution, so as to contain 0.064 Units/ml [standard dilution I] and 0.096 Units/ml [standard dilution II].

PROCEDURE FOR BIOASSAY OF INSULIN

Mice are divided into Four Groups each containing 25 Mice and Insulin is injected s.c. as Follows.

  MOUSE METHOD  
DOSEStandard 1
(0.25 ml)
Standard 2
(0.25 ml)
Test sample 1
(0.25 ml)
Test sample 2
(0.25 ml)
Convulsions0.50.511
Percentage
Convulsions
25 %25 %50 %50 %

Mice are put in an air incubator at 300 Degree Celsius and observed for one and a half hour.

Air Incubator consists of Glass front which as six shelves in it.

The Temperature is Thermostatically Controlled.

Two Mice are kept in each of the boxes made up of perforated sheets of metal.

The Mice which convulse or die are taken out of the incubator and observed.

These Mice usually convulse severely.

The mice when placed on its back and when it fails to upright itself should as well considered as convulsion.

Convulsion Mice can be saved by an Injection of 0.5 ml of 5% Dextrose solution.

Percentage convulsion produced by the test sample are compared with that of the standard sample.

Those animals which Survive May be used again for another Experiment.

This animals can be reused after an interval of one week.

RABBIT METHOD FOR BIOASSAY OF INSULIN

Selection of Rabbits

The Rabbits should be Healthy, Weighing about 1800-3000 grams.

 They should then be maintained on a Uniform Diet.

Before the experiment they are fasted for 18 Hours before Assay.

Water is also withdrawn during the fasting period of rabbits.

STANDARD AND SAMPLE DILUTIONS:

The dilutions are prepared freshly by diluting with normal Na-CL Solution so as to contain 1 unit/ml and 2 units/ml.

  12 RABBITS  
No of Rabbits3333
SampleStandard
Dilution
1
Standard
Dilution
2
TEST
Dilution
1
TEST
Dilution
2

DOSES:

The Dose which can produce suitable fall in Blood Sugar level is calculated for the standard.

PRINCIPLE:

The potency of a test sample is estimated by comparing the Hypoglycaemic Effect of the sample with that of the standard preparation of insulin.

Any other suitable method can also be used.

EXPERIMENTAL PROCEDURE

Animals are divided into Four Groups of Three Rabbits each.

The rabbit are then put into an Animal holder.

They should be handled with care to avoid excitement.

FIRST PART OF THE TEST

In this part a sample of blood is taken from the marginal EAR vein of each Rabbit.

Presence of Reducing Sugar is estimated per 100 ml of blood.

A suitable Chemical method is used.

This Concentration is called Initial Blood Sugar Level.

The Four Groups of Rabbits are then given SC injections of insulin as follows;

From Each rabbit, a sample of Blood is withdrawn up to 5 hours at the interval of 1 hour each.

The Blood sugar is determined again.

This is known as Final Blood Sugar Level.

CROSS OVER TEST (SECOND PART OF THE EXPERIMENT)

The same animals are used for the second part of the experiment.

The experiment is carried out after one week.

Again they are fasted and initial Blood Sugar id determined.

The Grouping is reversed, that is to say, those animals which received the standard sample are given test sample.

And those which received the test sample are given standard sample.

Those animals which received the less dose of the standard are given higher dose of test sample and vice versa.

This Test is known as ‘TWIN CROSS OVER TEST’.

Mean percentage decrease in blood sugar of the first and second part is calculated.

Sr.noPharmacological EffectExample
1MydriaticsAtropine
2Anti-InflammatoryHydrocortisone
3Mioticsphysostigmine
4Anti-MicrobialChloramphenicol

RAT EPIDIDYMAL FAT-PAD METHOD:

In this method the ability of insulin to increase CO2 production by the fat-pad is taken as a parameter for the measurement of the potency of the insulin preparation.

RAT DAIPHRAGM METHOD:

In this method increase in the glycogen content of the muscle or increase in glucose uptake by muscle in response to insulin is taken as the index of the potency of insulin.

RADIO IMMUNO ASSAY:

It is the estimation of the concentration of the substance in a unit quantity of preparation using radio labelled antigens.

The number of drugs are valued nowadays by radioimmunoassay methods because these methods are highly specific and highly sensitive.

The radioimmunoassay of insulin is based on the ability of human insulin (unlabelled) to display beef’s insulin (which may be labelled) from the binding sites (i.e. antibodies).

The method involves the following steps:

(1) Bovine insulin is injected into the sheep, after a week the serum containing antibodies produced against bovine insulin is collected form the blood of the sheep.

(2) The serum containing the antibodies is exposed to radio labelled  insulin and the bound vs. free ratio is determined.

(3) The mixture of labelled antigens-antibodies is then added in different test-tubes labelled as standard and test.

About 6 concentrations of the standards are taken.

They are then added to different tubes and the bound vs. free ratio is again determined using gamma-counter.

(4) Standard curves are determined and the concentration of test insulin is determined using this standard curve.

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