LAB BASICS Endocrinology July 30 2008 Objectives for this topic To review how the laboratory can be...

LAB BASICSLAB BASICSEndocrinologyEndocrinology

July 30 2008July 30 2008

Objectives for this topicObjectives for this topic

•To review how the laboratory can be used in the assessment of a To review how the laboratory can be used in the assessment of a patient with endocrine abnormalities. patient with endocrine abnormalities. •To review some the basic chemical structures of major To review some the basic chemical structures of major hormones.hormones.•To review some principles of immunoassay methods used in To review some principles of immunoassay methods used in hormone measurements.hormone measurements.•To review what is available from the laboratory at LHSC and to To review what is available from the laboratory at LHSC and to understand some of the limitations of laboratory analyses.understand some of the limitations of laboratory analyses.

CHEMICAL MESSENGERSCHEMICAL MESSENGERS

Neurotransmitters Chemicals used to relay, amplify and modulate electrical

signals between a neuron and another cell. Synthesized endogenously i.e. within the presynaptic

neuronHormones 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.

CytokinesCytokines

• Cytokines that mediate natural immunityType 1 IFNTNFIL-1, IL-6Chemokines

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-12Migration inhibition factor (MIF)

Cytokines that stimulate HematopoiesisC-Kit LigandIL-3. IL-7GM-CSF, M-CSF, G-CSF, other CSF’s (colony stimulating factors)Eythropoietin

NeurotransmittersNeurotransmittersTransmitter Molecule Derived From Site of Synthesis

Acetylcholine Choline CNS, parasympathetic nerves

Serotonin5-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 sympathetic, sensory and enteric nerves

NITRIC OXIDE, NO Arginine CNS, gastrointestinal tract

The sites of the principal endocrine glandsThe sites of the principal endocrine glands

Principal endocrine glands of the human Principal endocrine glands of the human body, their secretion, function and typebody, their secretion, function and type

Transport of HormonesTransport 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 SystemControl of Endocrine System

Simple - self limiting signal, effect is self limitedMay 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 SystemDisorders 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 HORMONESECTOPIC HORMONES

• These are hormones or hormone-like products, produced by tumors located outside of the gland that normally produces them.

Biochemical structure of hormonesBiochemical 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 canexist in different molecular forms.Not all forms are biologically active

MEASUREMENT OF HORMONE MEASUREMENT OF HORMONE CONCENTRATIONSCONCENTRATIONS

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 MEASUREMENT OF HORMONE CONCENTRATIONSCONCENTRATIONS

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 IMMUNOASSAYCOMPETITIVE IMMUNOASSAY

Most hormones are measured by Most hormones are measured by immunoassay 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).


• 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 radio immunoassay – standard curve.

• Ideal RIA curve non-ideal RIA curve

• The label in the bound fraction is inversely proportional to the concentration of analyte.

uE3 calibration Second Trimester

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Estriol in first trimester

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• 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 ImmunoassaysNon-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 NON-COMPETITIVE IMMUNOASSAYSIMMUNOASSAYS

• 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 immunoassayNon-competitive immunoassay

Non-competitive immunoassayNon-competitive immunoassay

HCG assay by two site Fluoroimmunometric assay

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NON-COMPETITIVE NON-COMPETITIVE IMMUNOASSAY(SANDWICH TYPE)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 labelsImmunoassay 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 LLSGEndocrine 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”.


• 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 AnalyzerThe Siemens Centaur Analyzer


• 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


• 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


• 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 analyzerImmulite 2000 analyzer


• 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)


• 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)


• 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


• 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


• 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 ??


• 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


• 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


• SKIN– 25 hydroxy Vitamin D under the influence of

sunlight. Laboratory assay has to be able to quantitate equally both D2 and D3 forms.

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