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

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

5. 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

7. Radioimmunoassay - competitive binding
unlabelled hormone
labelled hormone AB

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

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

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

13. 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
Keq [BG]
Changes in binding proteins alter concentration of total and free hormone
Does ratio change in capillary bed?

14. 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

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

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

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

18. 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

19. 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

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

21. 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)

22. Feedback control of thyroid hormone synthesis and release

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

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

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

26. 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

27. 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

28. 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)