BIO 5406 Notes,
1/18/05
EXPERIMENTAL TECHNIQUES IN
ENDOCRINOLOGY
I. Incentives for Endocrine Research. [Hadley, pp. 38-40]
A. Gland without a function.
1. Adrenal glands first described -- 1563.
2. Essential
for life -- 1856.
3. Functions were
identified beginning 1895.
B. Extract without a function.
1. Inhibin, secreted by gonads, discovered in 1932.
2. Function as an inhibitor of FSH secretion was not discovered until
1975.
C. Observation of a specific illness or syndrome in humans.
1. "Experiments of nature."
2. Addison described signs and symptoms of adrenal
insufficiency in
man -- 1855.
D. Discovery of novel pharmaceutical agents.
1. Leptin for treatment of obesity.
E. Study of the adaptive features of endocrine mechanisms.
1. How do these mechanisms make a species more fit in its
environment?
2. Comparative endocrinology.
II. Importance of Animal Studies. [Hadley, pp. 3, 54-56]
A. Experimental diabetes in pancreatectomized dogs led to discovery
of insulin.
III. The Classic Technique of Endocrinology. [Hadley, pp. 41-43]
A. Procedure.
1. Remove gland (ex. castration).
2. Observe effects of removal.
3. Replace the putative hormone.
4. Observe effects of replacement.
B. Types of hormone replacement.
1. Implant of gland (ex. Berthold's expt).
2. Injection of crude extract.
3. Injection of purified extract.
4. Injection of synthetic hormone.
IV. Assay Systems.
A. Purpose is identification and/or quantitation of an unknown sample of
hormone.
B. Criteria to consider in an assay system.
1. Sensitivity.
2. Specificity.
3. Accuracy.
4. Ease of operation.
V. Bioassay. [Hadley, pp. 43-45]
A. Quantitative testing of a biological effect of a hormone preparation in
a living system.
B. Used to identify and measure the active principle in a body fluid or
tissue extract.
C. Effect of an unknown quantity of hormone is compared to the effect of
a standard preparation of the hormone.
D. Establish a simple, quantitative response system in which to test
extracts.
1. Does not necessarily involve the same species (ex. pigeon crop sac
bioassay can be used to measure mammalian prolactin).
E. Examples (figure).
F. Characteristics.
1. Very sensitive.
a. Must be able to demonstrate a dose-response relationship.
b. Important to work in proper dose range.
2. Not always specific (ex. both oxytocin and acetylcholine can cause
uterine
contractions).
a. Measure of biological activity, not chemical identity.
b. Multiple assays helpful.
3. Accurate.
4. Tedious and time-consuming.
VI. Competitive Protein Binding Assays. [Hadley, pg. 46, Yalow papera]
A. Based on competition for protein binding sites.
B. Procedure.
1. Produce a pure compound and label it with a radioactive tag
(ex. I125-insulin).
2. Select a protein to which the hormone will bind.
a. Hormone receptor.
b. Antibody.
c. Transport protein (ex. SHBG).
3. Incubate labeled hormone with binding protein.
H* + P H*- P
a. Separate bound hormone from free hormone.
1. Filter or centrifuge with absorbant charcoal.
b. Measure radioactive decay using liquid scintillation spectrometry.
4. Incubate labeled and unlabeled hormone together with binding
protein.
a. They will interact identically with the binding protein.
H
+
H* + P
H*-
P
H - P
5. Increase unlabeled hormone in incubation mixture ---->
decrease labeled hormone bound to protein.
6. Using a range of known concentrations of pure hormone, establish
a
standard curve.
Bound H*
Free H*
[Hormone]
7. Compare unknown to this standard curve.
C. Radioimmunoassay (RIA).
1. Binding protein is an antibody.
2. First RIA -- insulin.
a. Rosalyn
Yalow and Solomon Berson.
b. 1977 Nobel
Prize for Yalow.
3. Procedure.
a. Use purified hormone as an antigen to generate antibodies in an
animal (ex. rabbit).
1. More recently -- monoclonal antibodies.
b. Incubate labeled and unlabeled hormone with antibodies (figure).
4. Characteristics.
a. Very sensitive.
b. Very specific.
1. Identifies specific chemical structure, not biological activity.
2. Ex. gastrin and cholecystokinin cross-react.
c. Accurate.
d. Relatively easy to perform.
aYalow, R.S. Principles and clinical applications of radioimmunoassay. Modern Medicine
57(3):
68-72, 1989.
VII. Immunoassay. [Witherspoon paperb]
A. Based on binding of antibodies to the desired hormone.
B. Antibodies are linked to another chemical which allows easy
identification or quantitation of the bound antibody (figure).
1. Radiolabeled antibody.
2. Fluorescent label.
3. Luminescent label.
4. Colorimetric label.
C. Amount
of bound antibody is proportional to amount of ligand present.
1. Not competitive.
D. Home pregnancy test -- enzyme-linked colorimetric immunoassay
(figure).
1. Goal is to identify presence of human chorionic gonadotropin
(hCG) in a urine sample.
2. Dipstick contains antibodies to hCG (figure).
a. Antibodies also linked with a colorimetric reagent.
3. Hormone-bound antibodies are transported to a window on the
dipstick where they come into contact with a second set of anti-hCG
antibodies.
a. These antibodies are linked with an enzyme.
4. Sandwich is formed with hormone and two antibodies.
5. On contact, enzyme on one antibody causes color change in the
reagent linked to the other antibody.
6. Color change indicates positive pregnancy test.
bWitherspoon, L. Immunoassay: The evolution of a revolution in disease diagnosis.
Modern Medicine 57(3): 68-72, 1989.
VIII. Immunocytochemistry. [Hadley, pp. 40-41, 51-52]
A. Utilizes antibodies to specific hormones attached to fluorescent or
colored dyes.
B. Applied
to tissue sections to identify hormone-producing cells
(ex. insulin antibodies used to detect
beta cells in pancreas).
C. With electron microscopy, organelles involved in storage of hormones
can be located.
IX. Recombinant DNA Technology. [Hadley, pg. 52]
A. Insertion of gene for human hormone into bacteria.
B. Bacteria synthesize the hormone in large quantities.
C. Ex. recombinant human insulin.
1. Gene for human proinsulin inserted in E. coli.
2. Proinsulin produced in large quantities.
3. C-peptide is chemically cleaved and insulin is purified.
X. Transgenic Animals. [Hadley, pp. 52-54]
A. Introduce foreign DNA into animals early in embryonic development.
B. Produce animal models of endocrine diseases.
1. Gigantism produced in mice by inserting gene for rat growth hormone
into pronuclei of fertilized mouse eggs (fig. 3.7).
XI. Other Molecular Techniques.
A. Use of complementary DNA sequences to determine primary structure
of peptide or hormone receptors.
B. Cloning of genes responsible for peptide hormone synthesis or hormone
receptors.