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

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

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

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

Radioimmunoassay - competitive binding unlabelled hormone labelled hormone AB

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

Enzyme linked immunoabsorbant assay (ELISA) hormone + substrate ENZYME

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

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?

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

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

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

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

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

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

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

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)

Feedback control of thyroid hormone synthesis and release

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

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

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

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

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

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)