1. MECHANISM OF ACTION OF HORMONES
AND
ASSAY OF HORMONES.
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2. MECHANISM OF ACTION OF HORMONES
 Hormones are classified in several ways, one of which is
according to their mechanism of action. The basis of this
classification is dependent on their solubility either in
lipid or water. They are classified as follows:
I. Lipophilic (lipid-soluble)
II. Hydrophilic (water-soluble)
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3.  Lipophilic hormones get transported to their target cell by
associating with carrier proteins in the plasma since they
cannot dissolve in plasma. This process circumvents the
process of solubility while it also prolong the half-life of the
hormones. Their lipophilic nature makes it easy for them to
diffuse across the plasma membrane of the target cell to bind
with their specific receptors in the cytoplasm or nucleus.
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5.  Lipophilic hormones, which include steroid hormones,
iodothyronines, retinoids and calcitriols are relatively
small molecules (300-800 Da). With the exception of the
iodothyronines, they are not stored by hormone-forming
cells, but are released immediately after being
synthesized. Via intracellular receptors, they mainly act
on transcription. They diffuse across the cell membrane
and bind with their receptor in the cytoplasm (steroids)
or in the nucleus (thyroid hormones).
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7.  Hydrophilic hormones get transported to their target
site freely in the plasma since they are soluble in it. On
getting to the target cell, they are unable to pass
through the plasma membrane because of their
hydrophilic nature which contrasts with the
hydrophobic nature of the membrane interior. As a
result of this, there is need for a second messenger that
will relate the message carried by the hormone to the
cell.
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8.  The second messenger system is of three types:
 Adenylyl cyclase-cAMP second messenger system
 Phospholipid second messenger system
 Calcium-calmodulin second messenger system.
All these systems are utilized by hydrophilic hormones.
It should be noted that a specific hormone can utilize
more than just one of these systems in its action.
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10. GROUP I GROUP II
Steroids,
Polypeptides, proteins,
iodothyronines,
TYPES glycoproteins,
calcitriol,
catecholamines
retinoids
Solubility Lipophilic Hydrophilic
Transport
Yes No
proteins
Long (hours to
Plasma half-life Short (minutes)
days)
Receptor Intracellular Plasma membrane
cAMP, cGMP, Ca2+,
Receptor-
metabolites of complex
Mediator hormone
phosphoinositols,
complex
kinase cascades 10

11. ASSAY OF HORMONES
 Most hormones are present in the blood in extremely
minute quantities; some concentrations are as low as
one billionth of a milligram (1 picogram) per milliliter.
Therefore, it was very difficult to measure these concentrations by
the usual chemical means. An extremely sensitive method, however,
was developed about 40 years ago that revolutionized the
measurement of hormones, their precursors, and their metabolic
end products. This method is called radioimmunoassay.
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12. The method of performing radioimmunoassay is as follows:
 First, an antibody that is highly specific for the hormone to be
measured is produced.
 Second, a small quantity of this antibody is mixed with a
quantity fluid from the animal containing the hormone to be
measured and mixed simultaneously with an appropriate
amount of purified standard hormone that has been tagged with
a radioactive isotope.
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13. Note: There must be too little antibody to bind completely both
the radioactively tagged hormone and the hormone in the fluid to
be assayed. Therefore the natural hormone in the assay fluid and
the radioactive standard hormone compete for the binding sites of
the antibody. In the process of competing, the quantity of each of
the two hormones, the natural and the radioactive, that binds is
proportional to its concentration in the assay fluid.
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14.  Third, after binding has reached equilibrium, the antibody-
hormone complex is separated from the remainder of the
solution, and the quantity of radioactive hormone bound in this
complex is measured by radioactive counting techniques. If a
large amount of radioactive hormone has bound with the
antibody, it is clear that there was only a small amount of natural
hormone to compete with the radioactive hormone and therefore
the concentration of the natural hormone in the assayed fluid
was small.
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15. Conversely, if only a small amount of radioactive hormone has
bound, it is clear that there was a large amount of natural hormone
to compete for the binding sites.
 Another method for measuring the concentration of hormones in
the body is through the Enzyme-Linked Immunosorbent Assay
(ELISA). This method can be used to measure almost any protein
including hormones. This test combines the specificity of
antibodies with the sensitivity of simple enzyme assays. The
figure below shows the basic elements of this method.
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16. AB1 & AB2 are antibodies that recognize the hormone (H) at different binding
sites, and AB3 is an antibody that recognizes AB2. E is an enzyme linked to AB3
that catalyzes the formation of a coloured product (P) from a substrate (S). The
amount of the product is measured using optical methods and is proportional
to the amount of hormone in the well if there are excess antibodies in the well.
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17. This method is often performed using a plastic plate with 96 small
wells.
 Each well is coated with an antibody AB1 that is specific for the
hormone being assayed.
 Fluid containing the hormone is added to each of the wells
followed by a second antibody AB2 that is also specific for the
hormone but bind to a different site of the hormone molecule.
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18.  A third antibody is added that recognizes AB2 and is coupled to
an enzyme that converts a suitable substrate to a product that
can be easily detected by colorimetric or fluorescent optical
methods.
In contrast to competitive radioimmunoassay methods, ELISA
methods use excess antibodies so that all hormone molecules are
captured in antibody-hormone complexes. Therefore the amount of
hormone present in the sample is proportional to the amount of
product formed.
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19. The ELISA method has become widely used in clinical
laboratories because:
1. It does not employ radioactive isotopes
2. Much of the assay can be automated using 96-well plates
3. It has proved to be a cost-effective and accurate method
for assessing hormone levels.
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