Bioassay of insulin.pptx

Insulin bioassay is the measurement of insulin potency by biological activity measurement using a standard biological system. An often used technique is to inject insulin samples into animals, usually rats or rabbits, and then track their blood sugar levels to see how well insulin lowers blood sugar. An alternate strategy is to measure glucose absorption or metabolic reactions using cultured cells sensitive to insulin, including adipocytes or muscle cells. By giving important information on insulin potency, purity, and stability, these tests guarantee the quality of insulin products intended for therapeutic use. Bioassays continue to be necessary for insulin preparation quality control and regulatory clearance even with the development of analytical methods.

1. BIOASSAY OF INSULIN Presented by:- Lokesh Patil B.Pharm 5 th Sem, 3 rd Year

2. INTRODUCTION Insulin was discovered in 1921 by Sir Frederick G Banting, Charles H Best & JJR Macleod. Insulin is a peptide hormone produced by Beta cells of pancreatic islets (Islets of Langerhans) It is considered to be the main anabolic hormone of the body. The human insulin is composed of 51 Amino Acids. It is dimer of an A-chain & B-chain, which are linked together by disulfide bonds. A chain contains 21 amino acids & the B-chain contains 30 Amino Acids. Insulin is practically insoluble in water, it dissolves in dillute mineral acids.

3. Bioassay To compare the test substance with the international standard preparation. To find out how a test substance is required to produce the same biological effect as produced by the standard. Principle The potency of a test sample is estimated by comparing the hypoglycemic effect of the sample with that of the standard preparation of insulin.

4. Bioassay of insulin Standard preparation and unit :- It is pure, dry crystalline insulin. One unit contains 0.04082 mg. This 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 (60 micrograms) (0.2 ml) of insulin and dissolve it in normal saline. Acidify it with HCL to pH 2.5. Add 0.5% phenol as a preservative. Add 1.4% to 1.8% glycerin. Final volume should contain 20 units/ml. Store the solution in a cool and dry place .

5. Bioassay of insulin Preparation of test sample solution :- Accurately weigh 20 units (60 micrograms) (0.2 ml) of test sample and dissolve it in normal saline. Acidify it with HCL to pH 2.5. Add 0.5% phenol as a preservative. Add 1.4% to 1.8% glycerin. Final volume should contain 20 units/ml. Store the solution in a cool and dry place .

6. 1. Rabbit method Selection of rabbits :- Rabbits should be healthy, weighing about 1800-300 grams. They should maintain fast for 18 hours. Standard & sample dilution :- Should be freshly prepared by diluting with normal NaCl solution so as to contain 1 unit/ml & 2 units/ml. Doses: - The dose that can produce a suitable fall in blood sugar level is calculated. Experimental procedure :- Animals are divided into 4 groups of 3 rabbits each. The rabbits are then put into an animal holder They should be handled with care in order to avoid excitement.

7. First part of the test A blood sample is taken from each rabbit's marginal ear vein . The presence of reducing sugar is estimated per 100 ml of blood by a suitable chemical method. This concentration is called the “ initial blood sugar level ” The 4 groups of rabbits are then given subcutaneous injections of insulin as follows 12 Rabbits 3 Rabbits Test sample dilutions II 3 Rabbits Test sample dilution I 3 Rabbits Standard dilution II 3 Rabbits Standard dilutions I

8. Second Part of the test For the second part the same animals are used. The experiment can be carried out after one week. Again the rabbits are fasted and initial blood sugar is determined. The grouping is reversed, that is:- those animals that rec ei ved the standard are given the test and those that rec ei ved the test are now given the standard. Those animals that rec ei ved the lower dose of the standard are given the higher dose of the test sample and vice versa. This test is known as “ Twin Cross Over Test”.