Download Introduction to the endocrine system and the hypothalamic

LAB BASICS
Endocrinology
July 30 2008
Objectives for this topic
•To review how the laboratory can be used in the assessment of a
patient with endocrine abnormalities.
•To review some the basic chemical structures of major
hormones.
•To review some principles of immunoassay methods used in
hormone measurements.
•To review what is available from the laboratory at LHSC and to
understand some of the limitations of laboratory analyses.
CHEMICAL MESSENGERS
Neurotransmitters
 Chemicals used to relay, amplify and modulate electrical
signals between a neuron and another cell.
 Synthesized endogenously i.e. within the presynaptic
neuron
Hormones
 Chemical messengers from one organ (or group of cells)
to another.
Cytokines
 Peptides used as messengers between cells and for
controlling the inner environment at the cellular level.
Cytokines
• Cytokines that mediate natural immunity
Type 1 IFN
TNF
IL-1, IL-6
Chemokines
 Cytokines that regulate Lymphocyte activation, growth and
differentiation
IL-2, IL-4, TGFß
 Cytokines that regulate Immune-mediated Inflammation
IFNγ , Lymphotoxin, IL-5, IL-10, IL-12
Migration inhibition factor (MIF)
 Cytokines that stimulate Hematopoiesis
C-Kit Ligand
IL-3. IL-7
GM-CSF, M-CSF, G-CSF, other CSF’s (colony stimulating factors)
Eythropoietin
Neurotransmitters
Transmitter Molecule
Derived From
Site of Synthesis
Acetylcholine
Choline
CNS, parasympathetic nerves
Serotonin
5-Hydroxytryptamine (5-HT)
Tryptophan
CNS, chromaffin cells of the gut, enteric cells
GABA
Glutamate
CNS
Glutamate
CNS
Aspartate
CNS
Glycine
spinal cord
Histamine
Histidine
hypothalamus
EPINEPHRINE
Tyrosine
adrenal medulla, some CNS cells
NOREPINEPHRINE
Tyrosine
CNS, sympathetic nerves
DOPAMINE
Tyrosine
CNS
Adenosine
ATP
CNS, peripheral nerves
ATP
NITRIC OXIDE, NO
sympathetic, sensory and enteric nerves
Arginine
CNS, gastrointestinal tract
The sites of the principal endocrine glands
Principal endocrine glands of the human
body, their secretion, function and type
Transport of Hormones
 Peptide/Protein Hormones -hydrophilic, dissolve in water
 Steroid & Thyroid Hormones - require carrier proteins
•
•
•
•
CBG - cortisol, TBG - thyroid hormones, SHBG - sex hormones
Some are highly specific binding proteins
Albumin - low affinity, high capacity carrier
Equilibrium between bound and free forms of the hormone, only
the free form can bind to receptors
• The physiological state corresponds to the concentration of free
hormone
Control of Endocrine System
 Simple - self limiting signal, effect is self limited
May or may not have ‘threshold’ to limit noise
 Negative Feedback - signal induces response,
response inhibits production of signal
 Positive Feedback - response feeds back to
induce more signal. Amplification continues until
system can no longer respond, or until signal
removed
 Inhibitory Control - release of hormone control by
an inhibitor. Remove inhibitor to allow release
 Endocrine Rhythms – daily, monthly, seasonal
rhythms, external cues can modify rhythms
Disorders of the Endocrine System
 Oversecretion,
– loss of feedback control
– exogenous secretion (another, usually uncontrolled,
source - “ectopic”)
 Undersecretion
– loss of stimulation
– organ failure
 Disordered metabolic pathway
– reduced end product, increased intermediates (may have
endocrine activity), blocked from end product
 May affect growth, development, metabolism,
behaviour
ECTOPIC HORMONES
• These are hormones or hormone-like products,
produced by tumors located outside of the
gland that normally produces them.
Biochemical structure of hormones
•
•
Steroid Hormones
Lipid soluble
– Diffuse directly into cells
– Are derived from cholesterol
– EXAMPLE - CORTISOL
•
Non-steroid hormones
–
–
–
–
–
–
Water soluble
Receptors on the cell surface
Amines (Example catecholamines)
Peptides (eg. ACTH, ADH, Oxytocin)
Proteins (eg. GH, Insulin, Prolactin
Glycoproteins (eg. FSH, LH, TSH)
NOTE: Peptide hormones can
exist in different molecular forms.
Not all forms are biologically active
MEASUREMENT OF HORMONE
CONCENTRATIONS
Bioassays
 Measure biological response of hormone in vivo system,
tissue culture or whole animal
 Time consuming, technically demanding, poor accuracy
and precision
Chemical Reactions
 Obsolete
 Examples are PBI for thyroxine, fluorometric assays for
serum cortisol, urinary ketosteroids and ketogenic
steroids, Pisano reaction for VMA, HIAA and others
 Measures concentration or amount excreted, not activity
MEASUREMENT OF HORMONE
CONCENTRATIONS
Chromatography
 Works well for small molecules, especially those from the
adrenal medulla, example VMA, catecholamines
 HPLC introduced in 1976 for clinical applications
 LC/MS/MS becoming method of choice for small analytes
Immunoassays






Poly or monoclonal.
High throughput, accuracy and precision.
Measures concentration not activity.
Many hormones are measured by Immunoassay
Works best for larger molecules, especially proteins.
Specificity an issue when differentiating a hormone from
an inactive metabolite.
COMPETITIVE IMMUNOASSAY
Most hormones are measured by
immunoassay
• Basic Components of immunoassay
– The analyte to be measured.
– One or more antibodies or binding agent
specific to the analyte to be measured.
– A label (detection system).
COMPETITIVE IMMUNOASSAY
• The tracer molecule is attached to the antigen.
• After an incubation period where equilibrium is
obtained, an agent is added to separate the
bound from the free analyte.
• A pellet may be obtained by centrifugation that
contains only the bound fraction, the free fraction
is discarded.
• Because of the competition, the amount of tracer
in the pellet is inversely proportional to the
amount of analyte in the sample. The higher the
concentration in the sample the less of the tracer
will be able to bind to the binding agent.
COMPETITIVE IMMUNOASSAY
• Competitive radio immunoassay – standard curve.
Estriol in first trimester
uE3 calibration Second Trimester
120000
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Counts
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60000
40000
40000
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20000
0
0.00
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0
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Estriol Values
2nd trimester assay AutoDelfia
• Ideal RIA curve
non-ideal RIA curve
• The label in the bound fraction is inversely
proportional to the concentration of analyte.
6.00
COMPETITIVE IMMUNOASSAY
• Binding agents in a competitive
immunoassay may be:
– Second antibody (e.g. mouse anti-rabbit)
– Charcoal
– Biotin
– Coated tube technology
– Antibodies attached to magnetic beads
– Poly ethylene glycol (PEG) protein
precipitation
Non-competitive Immunoassays
• For competitive immunoassay the antibody
concentration is LIMITED.
• For non-competitive immunoassays the antibody
is in EXCESS. This means that all the analyte
will be bound to the antibody. Label is usually
on a second antibody. The label in the bound
fraction is directly proportional to the
concentration of the analyte to be measured.
NON-COMPETITIVE
IMMUNOASSAYS
• ALL OF THE ANALYTE BINDS TO AN
UNLIMITED CONCENTRATION OF
LABELLED ANTIBODY.
• THE BOUND FRACTION IS DIRECTLY
PROPORTIONAL TO THE
CONCENTRATION OF THE ANALYTE
Non-competitive immunoassay
Non-competitive immunoassay
HCG assay by two site
Fluoroimmunometric assay
5000000
Counts
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HCG KIU/L
15.00
NON-COMPETITIVE
IMMUNOASSAY(SANDWICH TYPE)
 No competition step in the procedure
 Uses larger amounts of Ab
 All the analyte will be bound to the antibody (solid
phase Ab).
 2nd Ab (tracer) binds to distinct epitope on analyte
 The label in the bound fraction (signal) is directly
proportional to the concentration of the analyte to be
measured.
 Better suited to monoclonal Ab
 Can be easily automated on large chemistry
platforms
 Includes ELISA type assays
Immunoassay labels
• Radioisotopes, such as I125, Co60, P32, H3 etc.
• Fluorophor labels such as Eu (europium chelates).
• Enzymes such as ALP, firefly luciferase, G6PDH that can
generate a colored complex if incubated with the correct
substrate following the separation step.
• Cofactors such as ATP, FAD.
• Free Radicals such as Nitroxide
• Inhibitors such as methotrexate
• Metals such as gold sol, selenium, silver sol.
• Particles such as bacteriophages, erythrocytes, latex
beads.
• Polynucleotides such as DNA
• Enzyme substrates such is Galactosyl-umbelliferone
Endocrine Laboratory LLSG
• Hypothalamus
– Secretes small labile peptides that cannot be
measured in peripheral blood.
– Cortrasyn, TRH, GnRH, GHRH are given to a patient
to measure pituitary response. (CIU consult)
– Note that many anti-psychotic drugs block dopamine
(aka PIF) secretion and lead to an increase in serum
prolactin levels. Psychiatrists often measure prolactin
level as “Therapeutic Drug Monitoring”.
Endocrine Laboratory LLSG
• Anterior Pituitary
–
–
–
–
–
–
–
TSH, measured daily on the Siemens Centaur
LH, Siemens Centaur
FSH, Siemens Centaur
GH, Immulite 2000 (Siemens)
ACTH, IRMA method from DiaSorin.
Prolactin, Siemens Centaur
HCG in post menopausal women (Core Lab)
The Siemens Centaur Analyzer
Endocrine Laboratory LLSG
• Posterior Pituitary
– Vasopressin (ADH) is measured by the Buhlmann RIA
double-antibody method. Method involves an
extraction and two 24 hour incubation stages. Test
performed about every two weeks with a reference
range of 0.8 to 3.5 pmol/L.
– Oxytocin: not available and limited clinical utility
Endocrine Laboratory LLSG
• Thyroid Gland - Cortex
–
–
–
–
–
–
–
–
Total T4 (obsolete)
Free T4 (Centaur Analyzer)
Total T3 (?)
Free T3 (Centaur Analyzer)
T3 uptake (obsolete)
PBI (obsolete)
Reverse T3
TBII, LATS
Endocrine Laboratory LLSG
• Thyroid Gland Cortical region
– Thyroglobulin (IRMA isotope kit from Kronus)
• Thyroid Gland medulla
– Calcitonin (Immulite)
• Thyroid Antibodies
– Anti-thyroglobulin (Anti-TG Abs – Immulite)
– Anti-thyroid peroxidase (Anti-TPO Abs - Immulite)
• Parathyroid Glands
– PTH (Immulite)
Immulite 2000 analyzer
Endocrine Laboratory LLSG
• Adrenal Gland - Cortex




Cortisol - plasma am
Cortisol - plasma pm
Urinary free cortisol – below 380 nmol/d
Urinary cortisol – below 789 nmol/d
• Urinary 17-OH corticosteroids by the Porter Silber method (phenylhydrazine)
or 17-Ketogenic steroids by the Zimmerman reaction, now obsolete.
 Aldosterone, DHEAS (on Immulite 2000)
• Urinary 17-Ketosteroids by the Zimmerman (M-dinitrobenzene) reaction,
now obsolete.
17 Hydroxy progesterone (RIA)
Endocrine Laboratory LLSG
• Adrenal Gland - Medulla
– Urine VMA total 24 hour or VMA per mmol/creat
– Urine HVA total 24 hour or HVA per mmol/creat
– Urinary and Plasma Catecholamines by HPLC
– Dopamine
– Norepinephrine
– Epinephrine
– Urine and Plasma Serotonin and Histamine by ELISA
– Urine Metanephrine by HPLC
– Plasma Metanephrine ???? (LC/MS/MS)
Endocrine Laboratory LLSG
• Stomach, Liver and GI
– Gastrin by RIA – I125 label is on the gastrin molecule
– Secretin not measured
– Ghrelin methods available for research – stimulates
GH secretion from the AP
– Insulin Like Growth Factor 1 or IGF-1. Measured on
the Immulite analyzer.
– HIAA by HPLC for diagnosis of Carcinoid
– Chromogranin A by ELISA
Endocrine Laboratory LLSG
• Pancreas
- Glucose in the Core laboratory
- Insulin by immunoassay on the Immulite analyzer
- C-peptide on the Immulite analyzer to determine
endogenous insulin secretion.
- Glucogon – not available
- Somatostatin (also from hypothalamus) – not available
Endocrine Laboratory LLSG
• Ovary and Placenta
-
HCG to Dx pregnancy and HCG for MSS
Estradiol on the Siemens Centaur
Progesterone on the Siemens Centaur
Androstenedione on the Siemens Immulite
-
Inhibin A
Inhibin B
Total inhibins
PAPP-A
Estriol
- Urinary 2/16 αEstrogen Metabolite Ratio ??
Endocrine Laboratory LLSG
• Testes
-
Total Testosterone on the Siemens Centaur
Free Testosterone by equilibrium Dialysis
Free Testosterone by analog method
Bioavailable Testosterone by calculation using Total
testosterone and SHBG.
- 5α-dihydrotestosterone (from testosterone) - ELISA
Endocrine Laboratory LLSG
• Kidney
– Renin by RIA
• Peripheral specimen may be supine or standing
• Renal vein sampling – need to work with CIU and DI to get
appropriate specimens
– EPO on the Immulite (cannot differentiate normal from recombant
forms)
– 1, 25 dihydroxy Vitamin D from 25 hydroxy Vitamin D
Endocrine Laboratory LLSG
• SKIN
– 25 hydroxy Vitamin D under the influence of
sunlight. Laboratory assay has to be able to
quantitate equally both D2 and D3 forms.
Endocrine Laboratory LLSG