Transcript Assessing endocrine function

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
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
+ Ab
H . Ab  H*. 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
AB
labelled
hormone
Immuno-radiometric assay (IRMA) double antibody
hormone
Radioactive label
Enzyme linked immunoabsorbant
assay (ELISA)
hormone
ENZYME
+
substrate
colour intensity
radioactivity
% bound radioactivity
Standard curves for different
competitive binding assays
hormone conc.
RIA
hormone conc.
hormone conc.
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
Phosphate
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
Graves’ disease measuring
antibodies
Patient’s serum (IgG)
Thyroid growth
TSH receptors
(porcine)
I125-TSH (bovine)
Assay of antibodies to the human TSH
receptors
Patients serum Add
125I-TSH
+ TSH
receptors
(bovine)
(porcine)
Precipitate Count
Feedback control of the H-P
axis
External
stimuli
Feed back
HYPOTHALAMUS
3o
PITUITARY GLAND
2o
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 High basal
response
Low basal hyper 1o
response
Pituitary function tests
Hypothalamus
Feed back
Bolus injection of releasing hormone
PITUITARY GLAND
Measure trophic hormone
Low or absent response
2o
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)