Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

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Chapter | 20 Thyroid Function
Specific antibodies have replaced the use of TBG so that
there is high specificity for the assay of proteins, polypeptides,
haptens, and most of the hormones. Polyclonal
antibodies give accurate results and are usually used for
these hormone assays. The mean normal T 4 -RIA in dogs
is 2.3 0.8 μ g/dl (29.6 10.3 nmol/l) with an observed
range of 0.6 to 3.6 μg/dl (7.7 to 46.3 nmol/l), which is comparable
to the 1.5 to 3.6 μg/dl (19.3 to 46.3 nmol/l) of Sims
et al. (1977) . T 4 -RIA is also widely used in cats because of
the high prevalence of hyperthyroidism in this species. The
reference range for cats is 0.1 to 2.5 μg/dl (1.3 to 32.3 nmol/
l). In the horse, T 4 -RIA is also quite low, 0.9 to 2.8 μg/dl
(11.6 to 36.0 nmol/l). Messer et al. (1995) reported a mean
T 4 -RIA of 21.42 3.46 nmol/l in 12 adult horses.
2 . Thyroxine by Enzyme-Labeled Immunoassay
An important advance in hormone assay is the development
of an enzyme-labeled immunoassay (EIA) test comparable
in every way to radioimmunoassay except that
the labeling is by an enzyme rather than radioiodine. This
method has some obvious advantages in that there is no
need to use radioactivity and the enzyme can be assayed
in any laboratory. One system labels T 4 with malate dehydrogenase
(MD), which in competition with unlabeled T 4 ,
binds to antibody. This method has the acronym EMIT for
enzyme multiplied immunosorbent test. This method has
another advantage in that the labeled and unlabeled fractions
need not be separated. The MD when bound to T 4
is inactive, and when it binds to the immunoglobulin, it is
activated. The activity of malate dehydrogenase is assayed
by standard enzyme methodology, and the T 4 is read from
a standard curve as in a radioimmunoassay.
Another system uses two separate recombinant fragments
of the enzyme β -galactosidase ( Horn et al., 1991 ).
The individual fragments, enzyme donor (ED) and enzyme
acceptor (EA), are inactive but when they recombine, they
form the active enzyme. Thyroxine is bound to ED. In the
presence of thyroxine antibody, the thyroxine-ED-antibody
complex inhibits recombination with EA, and no active
β -galactosidase is formed. When sample thyroxine and a standard
amount of thyroxine-ED are mixed, they compete for a
standard amount of antibodies. Thus, unbound thyroxine-ED
accumulates in direct proportion to the amount of sample
thyroxine. The unbound thyroxine-ED is free to bind with
EA to form the active β -galactosidase. In this case, enzyme
activity is directly proportional to the amount of thyroxine.
This method had a very high correlation coefficient when
compared to an RIA ( r 0.969), EIA (r 0.966) and a
fluorescence polarization immunoassay (FPIA, r 0.939).
Another variation of the nonisotopic immunoassay is
the fluorescence immunoassay in which a fluorochrome
is tagged to the T 4 . The T 4 concentration is inversely proportional
to the fluorescence as in the RIA. This is also a
sensitive test, but it requires a sensitive spectrofluorometer
for the assay.
Chemiluminescence is another nonisotopic method with
comparable accuracy ( Wilkinson et al., 1993 ). Enzyme
immunoassays have not replaced radioimmunoassays for
hormones, but the principle is now widely used for antigen
or antibody assays using horseradish peroxidase (HRP) as
the enzyme label. The procedure is popularly known as the
enzyme-linked immunosorbent assay or by its acronym,
ELISA. This procedure has been adapted for T 4 by labeling
T 4 with HRP, coupling the HRP to a dye that indicates
enzyme activity and hence T 4 concentration.
3 . Triiodothyronine by Radioimmunoassay
Triiodothyronine is also commonly assayed by radioimmunoassay
(T 3 -RIA). In the dog, the mean normal T 3 -RIA is
107 18 ng/dl (1.6 0.3 nmol/l) with an observed range
of 82 to 138 ng/dl (1.26 to 2.12 nmol/l) ( Kaneko, 1997 ). It
closely parallels T 4 -RIA in the dog so that the simultaneous
determination of T 4 -RIA and T 3 -RIA will increase the
diagnostic accuracy of either one alone. In cats, T 3 -RIA is
less widely used in comparison to T 4 -RIA. The reference
range for cats is 15 to 50 ng/dl (0.23 to 0.77 nmol/l). In
the horse, Messer et al. (1995) reported a mean T 3 -RIA of
0.85 0.52 nmol/l.
4 . “Free ” Thyroxine and “Free ” Triiodothyronine
“Free ” thyroxine (fT4 ) is the unbound fraction of the
total circulating T 4 , and its concentration is controlled by
the equilibrium between TBG and TBG-T 4 (Section III).
Equilibrium dialysis is now considered the best method
for determining the free hormones, but it is often too laborintensive
for use in many clinical laboratories. An equilibrium
dialysis method for free thyroxine is commercially
available so that this method is now widely used.
The fT 4 concentration for dogs is 0.52 to 2.7 ng/dl (6.7
to 34.7 pmol/l). In the hyperthyroid cat, Hays et al. (1988)
found no differences in dialyzable T 4 from the normal.
They inferred that the total T 4 is sufficient for diagnosis
and that the free hormone is not needed in the cat. In the
horse, Messer et al. (1995) reported a mean fT 4 -RIA of
14.0 1.16 pmol/l.
The fT 3 -RIA parallels fT 4 -RIA in its binding characteristics.
Because fT 3 is the physiologically active form
of the hormone, it is potentially the single most reliable
test of thyroid function. In the horse, Messer et al. (1995)
reported a mean fT 3 -RIA of 0.89 0.53 pmol/l.
5 . Thyroid-Binding Globulin, Thyroglobulin, and
Thyroid Autoantibodies
Thyroid-binding globulin (TBG) and thyroglobulin (Tg) or
colloid are measured by RIA. The standard technique for