endo 1.06 investigation and assessment of endocrine disease measuring hormones - bioassays,...

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

Cushing’s syndrome

Bioassays?

H . Ab H*. Ab

Concentration of radioactivity [ H*] is inversely proportional to the

concentration of the unlabled hormone in the sample or standard

H

H *+ Ab

Bioassay of LH by measuring its ability to stimulate

testosterone secretion in Leydig cells of the testis

Radioimmunoassay - competitive binding

unlabelled hormone

labelled hormone

AB

Immuno-radiometric assay (IRMA) - double antibody

Radioactive labelhormone

Enzyme linked immunoabsorbant assay (ELISA)

ENZYME + substrate

hormone

Standard curves for different competitive binding assays

hormone conc. hormone conc. hormone conc.

% b

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tyRIA IRMA ELISA

Bound versus free hormonesAn 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 Feedback control of the H-P axisaxis

HYPOTHALAMUS

PITUITARY GLAND

EFFECTOR ORGAN

Feed

back

External stimuli

3o

2o

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 GLAND

Measure trophic hormone

Feed

back

1o

Pituitary function tests

Injection of releasing hormone

HypothalamusHypothalamus

High basal response

hypo 1ohyperLow basal response

PITUITARY GLAND

Measure trophic hormone

Feed

back

Pituitary function tests

Bolus injection of releasing hormone

HypothalamusHypothalamus

Low or absent response

2o

PITUITARY GLAND

Measure trophic hormone

Feed

back

3o

Pituitary function tests

Bolus injection of releasing hormone

HypothalamusHypothalamus

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

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

Hypofunction of an endocrine organ - stimulation tests

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

Hyperfunction of an organ -

suppression test

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)

Hypofunction of an endocrine organ - stimulation tests