Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays,...
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![Page 1: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/1.jpg)
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
![Page 2: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/2.jpg)
Assessing endocrine function
• Bioassays
• Radioimmunoassays
• IRMA’s Antibodies
• ELISA’s
• Protein binding assays
• Receptor assays
![Page 3: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/3.jpg)
![Page 4: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/4.jpg)
Pan-hypopitutarism - presumed autoimmune
Cushing’s syndrome
Bioassays?
![Page 5: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/5.jpg)
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
![Page 6: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/6.jpg)
![Page 7: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/7.jpg)
Radioimmunoassay - competitive binding
unlabelled hormone
labelled hormone
AB
![Page 8: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/8.jpg)
![Page 9: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/9.jpg)
Immuno-radiometric assay (IRMA) - double antibody
Radioactive labelhormone
![Page 10: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/10.jpg)
Enzyme linked immunoabsorbant assay (ELISA)
ENZYME + substrate
hormone
![Page 11: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/11.jpg)
Standard curves for different competitive binding assays
hormone conc. hormone conc. hormone conc.
% b
oun
d r
adio
acti
vity
rad
ioac
tivi
ty
colo
ur
inte
nsi
tyRIA IRMA ELISA
![Page 12: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/12.jpg)
![Page 13: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/13.jpg)
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?
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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
![Page 15: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/15.jpg)
Graves’ disease - measuring antibodies
Patient’s serum (IgG)
TSH receptors (porcine)
I125-TSH (bovine)
Thyroid growth
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Assay of antibodies to the human TSH receptors
Patients serum Add Precipitate Count + TSH 125I-TSH
receptors (bovine) (porcine)
![Page 17: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/17.jpg)
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
![Page 18: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/18.jpg)
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
![Page 19: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/19.jpg)
PITUITARY GLAND
Measure trophic hormone
Feed
back
1o
Pituitary function tests
Injection of releasing hormone
HypothalamusHypothalamus
High basal response
hypo 1ohyperLow basal response
![Page 20: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/20.jpg)
PITUITARY GLAND
Measure trophic hormone
Feed
back
Pituitary function tests
Bolus injection of releasing hormone
HypothalamusHypothalamus
Low or absent response
2o
![Page 21: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/21.jpg)
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)
![Page 22: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/22.jpg)
Feedback control of thyroid hormone
synthesis and release
![Page 23: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/23.jpg)
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
![Page 24: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/24.jpg)
Dynamic tests of endocrine function
• Hyperfunction of an endocrine organ - suppresion tests
• Hypofunction of an endocrine organ - stimulation tests
![Page 25: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/25.jpg)
Examples of dynamic tests of endocrine function
• Pituitary function tests
• Dexamethasone suppression test
• Insulin stimulation of GH
![Page 26: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/26.jpg)
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
![Page 27: Endo 1.06 Investigation and assessment of endocrine disease Measuring hormones - bioassays, radioimmunoassays, IRMAs and ELISAs Measuring ‘free’ thyroxine.](https://reader033.fdocuments.in/reader033/viewer/2022051417/56649d945503460f94a7c493/html5/thumbnails/27.jpg)
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
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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