This document discusses various techniques for hormone assays, including biological assays, immunological techniques, competitive radioimmunoassays, ELISA, and cytochemical methods. It then covers the principles and mechanisms of hormone action, including receptor interactions, cAMP-mediated pathways, and transcription/translation effects. Finally, it outlines the regulation of hormone secretion, distinguishing between direct feedback control and nervous system control through the hypothalamus.

1. General principles of
endocrinology -II
Dr. Sai Sailesh Kumar G
Associate Professor
Department of Physiology
RDGMC

2. Hormone assays
 Biological assay of hormones
1. Solvent extraction
2. Chromatography
3. Molecular sieving
4. Immunological techniques
5. Competitive radio assay
6. ELISA
7. Cytochemical methods

3. Hormone assays
 Immunological techniques
1. Fluorescence method for catecholamines
2. Gas liquid Chromatography for steroid hormones
3. Use of radio active isotopes for thyroid hormones

4. Hormone assays
 Immunological techniques
1. Fluorescence method for catecholamines
2. Gas liquid Chromatography for steroid hormones
3. Use of radio active isotopes for thyroid hormones

5. Hormone assays
 Competitive radio assay
1. Radio immunoassay
2. Applied to all protein and peptide hormones
3. Can be detected in concentration of micro gram
or nano grams
4. Antibody as the binding protein

6. Radio immunoassay
 Principle
 Binding sites on antibody are saturated with radio
labeled hormone
 The competition between free and labeled hormone for
the binding sites
 If the concentration of free hormone more, it binds with
binding sites
 Ratio of bound labeled to free hormone
 Method has high sensitivity and high specificity

7. Radio im munoassay

8. ELISA
 Enzyme linked immunosorbent assay
 Basic principle to measure concentration of hormone
 AB1, AB2 are antibodies for hormone (H)
 AB3 is added to recognize AB
 AB3 is linked to an enzyme that converts substrate to
product
 that can be easily detectable by colorimetric or fluorescent
optical methods
 The amount of product is proportional to the amount of
hormone present in the sample

9. ELISA

10. ELISA
 Advantages
 Cost effective method
 Does not employ radio isotopes
 Accurate method to assess hormone levels

11. Cytochemical assays
 More sensitive than RIA
 More complicated
 Time consuming
 Very useful to measure basal levels of hormone
secretion

12. Mechanism of hormone action
 Only cells with receptors for a specific hormone
respond
 Those cells lacking the specific receptors are un
affected
 Receptors on cell membrane – external receptors
 Receptors in cytoplasm – internal receptors

13. Mechanism of hormone action
 Peptide hormones binds to receptors on cell
membrane
 Steroid hormones bind with receptors in the cyto-
plasm
 Thyroid hormones binds with nuclear receptors

14. Hormone receptor interaction
 Two mechanisms
1. cAMP medicated hormone activity
2. Transcription and translation effects

15. cAMP mediated hormone activity
 Proteins and poly peptide hormones
 Amino acid derivatives – catecholamines
 Some prostaglandlins
 Large in size so can not enter the cells
 Acts as first messenger
 Binds with receptors on cell surface
 Activates enzyme adynylil cyclase on inner surface
of membrane

16. cAMP mediated hormone activity
 Activates enzyme adynylil cyclase on inner surface
of membrane
 Increase in the formation of intra cellular cAMP
 cAMP acts as second messenger
 Converts inactive protein kinase to active form
 Protein kinase causes phosphorylation of proteins
 Brings different functions

17. cAMP mediated hormone activity
 Protein kinase causes phosphorylation of proteins
 Brings different functions
 Change in the membrane permeability
 Activation or inactivation of rate limiting enzymes
 Increase or decrease protein synthesis by action
on ribosomes
 Regulation of release of hormones

18. cAMP mediated hormone activity

19. Transcription Translation effect
 Lipid soluble hormones easily enter the cell
 Steroid hormones
 1,25 DHCC
 Thyroid hormones
 Binds with receptor in cytoplasm or in nucleus
 Hormone receptor complex
 Stimulates transcription ( formation of mRNA)

20. Transcription Translation effect
 Stimulates transcription ( formation of mRNA)
 mRNA enters cytoplasm
 Activated ribosomes
 Synthesis of specific proteins
 Translation
 These proteins may be enzymes, structural
proteins, receptor proteins or secretory proteins

21. Transcription Translation effect

22. Regulation of secretion of
hormones
 Two control mechanisms
 Direct control
 Nervous control

23. Direct action
 Some hormones are controlled by blood
concentrations of same hormones
 Negative feedback
 Eg: Thyroid hormones

25. Indirect action
 Controlled by CNS
 Especially hypothalamus
 Secretes stimulatory and inhibitory hormones
 Based on feedback signals
 Negative feed back – decreases secretion
 Positive feedback – increase secretion

26. THANK YOU