Methods in endocrinology

Methods in endocrinology

1. Areas of study

   • Glands
   • Target tissues/cells

2. General methods

   • Surgical manipulation - e.g. removal/replacement of a gland
     • Physiological alterations
       • In target tissue
         • Loss of activity
       • Chnages in blood composition?
     • e.g. castration or adrenalectomy
       • reversed by hormone replacement therapy
   • Tissue extract preparation
   • Histological methods - ID target cells (immunohistochemistry, staining)
     • Cytological characteristics
       • Hypertrophic - enlarged tissue (not number of cells)
       • Organelles - number present, size etc.
         • Dilated ER/golgi = increased activity
       • Atrophy (wasting of tissue)
       • Hyperplasia (increased number of cells)
       • Disease status
     • Stains (of chemical components in cell)
       • Glycoproteins
       • Glycogen
       • Lipid
       • Can indicate function
   • Gene expression
   • Pharmacological - mimic/inhibit activity
   • Anima models

3. General considerations

   • Source
   • Structure and synthesis
   • Control of secretion and mechanism
   • Circulation and metabolism
   • Actions and roles
   • Pathophysiological roles
   • Comparative endocrinology

4. Studying the endocrine control of a physiological process

   • For hormones secreted by a ductless gland
     • Remove the gland
       • observe effects
     • Is effect restored after gland extract?
     • Purified/synthetic hormone induec same effect?
     • Secretion - mechanism and control
     • ID receptors on target cells
       • Many receptors found - some are referred to as orphans as their ligands are not known
   • Gross observation of tissue
     • Anatomical location
     • Size
     • Vascularisation / innervation
     • Pathophysiologies - e.g. goitre - indicating a thyroid problem
     • Immunocytochemistry

5. Techniques

   • Immunocytochemistry
     • Can involve
       • Ab's
         • Very specific
         • For peptide/protein hormones
       • Conjugated
         • With fluorescent tags/enzymes that produce a coloured prduct
     • Method
       • Tissue sample prepared
         • Incubated in primary Ab for specfic cell protein
           • Incubated with secondary Ab carrying a fluorescent tag
       • Visualisation
         • Dependent on method
           • Immunofluorescence (with fluorescent Ab's)
           • Immunoperoxidase (if enzyme-conjugated)
   • Fluorescent in situ hybridisation (FISH)
     • Detects gene amplification using a fluorescently labeleld gene probe
       • Aids treatment selection in breast cancer (HER2 amplification? - herceptin appropriate?)
   • Radioisotopes
     • Considerations
       • Half life of isotope
       • Trace distribution throughout the body
         • e.g. using radioisotopic iodine in the diagnosis (and treatment) of hyperthyroidism
       • Autoradiography
         • Uptake and incorporation of isotope into cells metabolic pathways - e.g. nucleotide synthesis / glucose metabolism
       • Cellular site of action
         • Steroids (gene expression) - intracellular
         • peptide hormones - extracellular
   • Radioimmunoassay (RIA)
     • Sensitive and highly specific (due to use of Ab's)
     • In vitro assay used to determine levels of hormone in sample (e.g. blood)
     • Method
       • Known amount of antigen is made radioactive and mixed with known amount of Ab = hot antigen
         • Next a sample (from the patient) of unknown antigen concentration is added = cold antigen
           • The unbound cold antigen competes for the Ab and displaces bound hot antigen
             • the amount of displacement correlates to the amount of cold antigen in the sample
               • The solution containing the unbound antigens (both hot and cold) is then separated and its radioactivity measured (geiger counter)
                 • The level of radiation is proportionate to the amount of displaced radioactive antigen
                   • This can be compared to a standard curve of solution radiation against known amounts of cold antigen (done previously)
     • Can be affected by episodic/diurnal release
   • Tissue extracts
     • Crude extraxcts (preparation from tissue - not purified)
       • Replace excised tissue
       • Can be purified - i.e. hormonal supplements
         • Can be used as treatment (i.e. insulin)
       • Tissue extract can be analysed using subcellular fractionisation
         • Using differential centrifugation
           • Subcellular fractions separated based on density/size
             • Then tested for receptor/hormone of interest (determines cellular location)
   • Detecting protein or mRNA of interest
     • Nothern blot
       • Detects mRNA
         • Simliar to western blot
           • Radiolabelled/fluorescent RNA primer used intead of Ab
     • Western blot
       • Detects proteins
         • Extract all proteins (lysis)
           • give negative charge or denature
             • Run on SDS page
               • Produce Ab
                 • Transfer SDS page to nitrocellulose
                   • Incubate with primary antibody
                     • Wash off excess Ab and incubate with secondary Ab (+ fluorescent tag)
                       • Detect fluorescence
   • PCR
     • Amplification of short mRNA sequence (testing transcription level)
       • Convert mRNA from cell into cDNA using a reverse polymerase
   • Bioassays
     • Use of a biological response to detect presence of a hormone in serum/tissue extract
     • The level of response to a hormone can be measured and a standard produced for known concetrations of hormone
       • Patient serum containing an unknown concentration of the hormone can then be tested and compared against the standard curve
     • Structure-activity assays
       • Looking at the structure of a compound and comparing this to its funtion
         • Antagonist or agonist?
           • Use known amounts of a hormone to produce a dose response curve (standard curve) - forms a sigmoid curve due to ligand receptor kinetics
             • Repeat with test compound
               • Is max response reached quicker/at all?
     • Site-directed mutagensis
       • Induce genetic mutation
         • To detect functional domains
           • Can be tested for using a reporter assay
             • Constructed gene with a hormone response element, basic promotor region and reporter gene (e.g. beta-galactosidase or luciferase)
               • If mutation affects a functional domain of the hormone/hormone receptor (e.g. steroid hormone receptor) then there is no trasncription of the reporter
                 • Visual result: beta-galactosidase expressing cells grow blue on X-gal medium; and luciferase produces light