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Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs a

Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine and thyroid hormone antibodies Negative feedback and interpreting results Dynamic tests of endocrine function

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Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs a

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  1. Endo 1.06 Investigation and assessment of endocrine disease • Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs • Measuring ‘free’ thyroxine and thyroid hormone antibodies • Negative feedback and interpreting results • Dynamic tests of endocrine function • Pituitary function tests • Dexamethasone suppression test • Insulin induced hypoglycaemia for GH stimulation

  2. Assessing endocrine function • Bioassays • Radioimmunoassays • IRMA’s Antibodies • ELISA’s • Protein binding assays • Receptor assays

  3. Bioassays? Pan-hypopitutarism - presumed autoimmune Cushing’s syndrome

  4. Bioassay of LH by measuring its ability to stimulate testosterone secretion in Leydig cells of the testis H H . Ab H*. Ab + Ab H * Concentration of radioactivity [ H*] is inversely proportional to the concentration of the unlabled hormone in the sample or standard

  5. Radioimmunoassay - competitive binding unlabelled hormone labelled hormone AB

  6. Immuno-radiometric assay (IRMA) - double antibody hormone Radioactive label

  7. Enzyme linked immunoabsorbant assay (ELISA) hormone + substrate ENZYME

  8. Standard curves for different competitive binding assays radioactivity colour intensity % bound radioactivity hormone conc. hormone conc. hormone conc. RIA ELISA IRMA

  9. Bound versus free hormones An equilbrium exists between bound and free hormone. H + BG H.BG Keq = [H.BG] (H bound) [H] [BG] (H free) [H] = [H.BG] x 1 Keq [BG] Changes in binding proteins alter concentration of total and free hormone Does ratio change in capillary bed?

  10. Measuring ‘free’ T4 1) Add serum sample or standard (T4 ) to latex beads coated with Abs 2) Add T4 labelled with alkaline phosphatase. This will bind to any unoccupied Ab binding sites 3) Add a substrate that will fluoresce when the alkaline phosphatase removes a phosphate group 4) Fluorescent intensity will be inversely proportional to the concentration of T4 Phosphate

  11. Graves’ disease - measuring antibodies Patient’s serum (IgG) TSH receptors (porcine) I125-TSH (bovine) Thyroid growth

  12. Assay of antibodies to the human TSH receptors Patients serum Add Precipitate Count + TSH 125I-TSH receptors (bovine) (porcine)

  13. Feedback control of the H-P axis External stimuli HYPOTHALAMUS 3o Feed back 2o PITUITARY GLAND EFFECTOR ORGAN 1o

  14. Endocrine disorders • 1o excess: low trophic hormone • 1o deficiency:high trophic hormone • 2o excess: high trophic hormone and hormones of target gland • 2o deficiency: low trophic hormone and hormones of the target gland • 3o deficiency: also low trophic hormone and hormones of the target gland

  15. Pituitary function tests Hypothalamus Feed back Injection of releasing hormone PITUITARY GLAND Measure trophic hormone hypo 1o hyper 1o High basal response Low basal response

  16. Pituitary function tests Hypothalamus Feed back Bolus injection of releasing hormone PITUITARY GLAND 2o Measure trophic hormone Low or absent response

  17. Pituitary function tests Hypothalamus 3o Feed back Bolus injection of releasing hormone PITUITARY GLAND Measure trophic hormone No response or delayed peak response (60 mins vs 20 mins)

  18. Feedback control of thyroid hormone synthesis and release

  19. Measuring trophic hormones and hormones of the peripheral endocrine gland High TSH - Low T3/T4 1o Hypothyroidism Low TSH - High T3/T4 1oHyperthyroidism Low TSH - Low T3/T4 2oHypothyroidism Other signs and symtoms usually determine whether primary (1o) or secondary (2o) e.g. goitre

  20. Dynamic tests of endocrine function • Hyperfunction of an endocrine organ - suppresion tests • Hypofunction of an endocrine organ - stimulation tests

  21. Examples of dynamic tests of endocrine function • Pituitary function tests • Dexamethasone suppression test • Insulin stimulation of GH

  22. Hypofunction of an endocrine organ - stimulation tests Example of a pituitary function test The ACTH response to a bolus injection of CRH is measured The grey shaded area shows the range of responses measured in control subjects In hypopituitarism there is no response In primary hypoadrenalism there is no feedback and basal ACTH concentrations are high

  23. Hyperfunction of an organ - suppression test Circadian rhythm of cortisol secretion The dexamethasone suppression test. In a normal person dexamethasone will suppress ACTH secretion (feedback) and cortisol production is consequently reduced. In pituitary- dependent Cushings only high doses may suppress ACTH secretion

  24. Hypofunction of an endocrine organ - stimulation tests Insulin induced hypoglycaemia to investigate suspected GH deficiency. Insulin decreases plasma glucose concentrations and in a normal person this stimulates the release of GH (A) A reduced or absent response is seen in a GH deficient patient(B)

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