Note: Descriptions are shown in the official language in which they were submitted.
1()35~5
To diagnose thyroid disfunction and evaluate thyroid therapy,
clinicians rely on tests which measure a patient's serum triiodithyronine
(T3) uptake level as well as tests which do quantitate the thyroxine (T4)
concentration. T3 uptake levels are determined in serum using T3 uptake
tests (e.g., Triosorb*); serum T4 levels are quantitated using tests which
measure total serum T4 concentration (e.g. T4 radioimmunoassay, Tetrasorb*).
Tests measuring total serum T4 are dependent upon the actual thyroid hormone
concentration present in the serum, whereas tests which measure T3 uptake
depend upon the thyroxine-binding protein (TBP) concentration in the serum
as well as the concentration of thyroid hormone. Both tests are important
in forming a clear understanding of the patient's thyroid stateO
Circulating T3 and T4 are bound to several constituents of the
blood of which the thyroxine binding globulin (TBG~ fraction contains the
major binding sites which are fixed in number. The binding of T3 and T4 on
the TBG molecule is one of competitive protein binding, that is, unbound T4
will replace T3 and T4 already on the molecule.
As there is only 1-2 mg of TBG/100 ml normal serum, the binding
sites, equivalent to approximately 20 micrograms of T4 per 100 ml of normal
serum, are readily saturated by a small increase of T4 concentration.
In hyperthyroidism, the binding sites on the TBG molecules are
nearly saturated with T3 and T4; in hypothyroidism, the binding sites are
highly unsaturated, resulting in an increased ability for T3 uptake by the
TBG molecule in the serum.
The Triosorb* assay and other similar tests for T3 uptake operate
on the principle of competitive protein binding. Serum T3 and T4 are primarily
bound to the binding sites on the TBG molecule. The number of unoccupied
binding sites is determined by the addition of radioactive T3 (T3*) to serum
in the presence of an adsorbing agent. When T3* is added to serum, any excess
not bound to the binding sites of the TBG molecule in the serum will be ad-
sorbed onto the added agentO Resin sponges are the adsorbing agents for the
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Triosorb test. For example, in hyperthyroidism, most of the TBG binding sites
are occupied by T3 and T4 and thus the added T3* will not be taken up by the
endogenous TBG molecules, but will be taken up by the test adsorbent. In
hypothyroidism, the reverse is true. Thus~ the amount of radioactive T3 bound
by the adsorbent directly reflects the thyroid state of the patient.
The tetrasorb assay for T4 is also based upon the principle of
competitlve protein binding. To determine the T4 concentration, T4 is extracted
from the serum releasing it from its binding protein, TBG. The serum extract
is then added to a solution containing a limited quantity of exogenous TBG to
which is bound radioactive T4 (T4*). A displacement reaction occurs in which
the T4 in the extract displaces the T4* from the exogenous TBG. This dis-
placed T4~ is then adsorbed onto the resin sponge used as the adsorbing agent.
As more T4 is added, more T4* is displaced from the TBG. The amount Or T4*
displaced from the TBG is therefore directly proportional to the amount of T4
present in the serum extract. In hyperthyroidism there is more T4 available
in the serum extract to displace T4~ from exogenous TBG than in euthyroidism
or hypothyroidism.
When normal levels of TBG are present in serum, these types of
thyroid function tests reflect the actual state of the patient. When TBG and
T4 levels are elevated, as in pregnancy or following estrogen ingestion in the
form of oral contraceptives, T3 uptake tests indicate the patient to be hypo-
thyroid. However, tests measuring total T4 indicate an euthyroid or sometimes
a hyperthyroid condition. Si~lply stated, pregnancy or estrogen ingestion re-
sults in an increase in the number of TBG molecules causing an increase in
hormone binding sites with a concurrent rise in thyroid hormone levels.
Thyroid function tests carried out on these sera at this time show an increased
T3 uptake, indicative of hypothyroid function and an increased T4 level indi-
cative of hyperthyroid function.
This invention seeks to provide a control serum prepared from
these types of sera and normal serum that will serve as a hypothyroid control
serum for T4 tests as well as T3 uptake tests. By normal serum Applicants
mean serum havi~ norm~l T3 and T4 values and which may be human, beef, sheep,
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goat, or other animal serum. Only horse serum has been found to lack utility.
In à first aspect this invention seeks to provide a method for ob-
taining a hypothyroid serum control comprising the following steps:
A. adding neutral, decolorizing carbon to blood serum in an amount
of about 5-20 percent based upon weight of carbon to volume of serum;
B. mixing the above mixture for about 24 hours at a temperature of
about 4C;
C. twice centrifuging the resulting slurry at 34,800 xg and at a
temperature of about 4C;
D. filtering the resultant supernatant;
E. lyophilizing the supernatant so obtained.
In a second aspect this invention seeks to provide in clinical chemis-
try procedures measuring a person's serum triiodothyronine uptake level and
thyroxine concentration, the improvement which comprises the use of a single
control serum standard as a hypothyroid reference control serum for both the
triiodothyronine uptake and thyroxine concentration tests, in which the hypo-
thyroid reference control serum is prepared by the following steps:
A. adding neutral, decolorizing carbon to blood serum in an amount
of about 5-20 percent based upon weight of carbon to volume of serum;
B. mixing the above mixture at a temperature of about 4c;
C. twice centrifuging the resulting slurry at 34,800 xg and at a
temperature of about 4c;
D. filtering the resultant supernatant;
E. lyophilizing the supernatant so obtained.
A serum is hyperthyroid by the tetrasorb and triosorb tests if the
percentage of T3 uptake is greater than 35, and the T4 concentration is above
14.5 mcg per 100 ml of serum. A serum is euthyroid if the percentage of T3 up-
take is 25 to 35 and the T4 concentration is 5.3-14.5 mcg per 100 ml of serum.
Hypothyroid serum has a T3 uptake of below 25 percent and less than 5 mcg of
T4 per 100 mls of serum.
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10391~35
Removing T3 and T4 from normal serum or serum containing elevated
TBG levels, such as by the methods outlined below, results in a serum judged
hypothyroid both by methods which measure T4 concentration and T3 uptake.
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EXAMPLE 1
Ingredients Quantity for 990 m1 ~420 vials)
lo Fresh, Normal Serum 2700 ml
20 Norit A*, Neutral Decolorizing480 g
Carbon pharmaceutical grade,
~Amend Drug CoO, Irvington, N.J.)
3. Water, Purified 560 ml
Method
1. Add Item #2 to 2400 ml of Item #1 in a 4 liter Erlenmeyer flask (about
20% carbon weight to volume of serum).
2. Swirl gently at room temperature until the charcoal particules are
dispersed in the liquid.
3. Place the mixture at 4C. Stir very gently for about 24 hoursO
4. At the end of 24 hours centrifuge mixture at high speed in a refrigerated
centrifuge ~34,800 x g)O
- 5. Decant the supernatant and centrifuge it again as in step 4.
6. After the second centrifugation, again decant the supernatant and
filter it by vacuum through a millipore* filter~
7. Pour the filtrate (1200 ml) into a suitable glass tray for lyophiliz-
ation and lyophilize.
8. Transfer the lyophilized material to a 2 liter Erlenmeyer flask and
add Item #3.
9. Let the mixture stand for 30 minutes, then aid the dissolving process
by swirling gently.
10. Add 300 ml of Item #l to the serum mixture obtained in step 9.
11. Dispense 2.14 cc into a vial that will hold a 2 ml fill and lyophilize.
Vials are reconstituted with 2.0 ml deionized distilled water. This serum
is used for a hypothyroid control in conjunction with all thyroid function
tests, i.e., the resulting serum control should have a T3 uptake level less
than 25% by the Triosorb method and a T4 concentration less than 5.3 mg/100
ml by the Tetrasorb methodO
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Modifications in Example 1 are of course possible. Centrifugation
(steps 4 and 5) may be eliminated; the millipore filter (step 6) may, of
course, be an Ertel apparatus; the serum may be human or animal; the require-
ment to dispense the mixtures (step 11) may, of course, be modified or done
away with completely; the time of mixing (step 3) may be shortened or length-
ened over a range of 3-30 hours; the percentage of carbon added may range
from 5-20 percent, with a range of about 10 to about 20 percent being pre-
ferred.
This procedure removes over 99% of the T3 and T4 from the starting
serum, effectively producing a T3 and T4-free serum while not significantly
affecting the total protein concentration, pH, or T4 binding capacity of
the serum.
The following example outlines the method used to remove T3 and
T4 from serum containing elevated TBG levels. Modifications of this example
s;m;lar to those outlined for Example l are also possible and encompassed by
this invention.
1()3~1~35
EXAMPLE 2
Ingredients Quantity for 900 ml (420 vials~
1. Serum containing a high thyroxine 1100 ml
binding globulin concentration
2. Neutral Decolorizing Carbon, 220 g
pharmaceutical grade
3. Fresh, normal serum 200 ml
Method
1. Add Item 2 to Item 1 in a 2 liter Erlenmeyer flask (about 20% carbon
weight to volume of serum).
2. Swirl gently at room temperature for one minute so that charcoal particles
are dispersed in liquid.
3. Place mixture at 4C and stir very gently for about 24 hours.
4. At the end of 24 hours, centrifuge mixture at high speed in a refrigerated
centrifuge (34,800 ~ g).
5. Decant the supernatant and centrifuge it again as in Step 4.
6. After second centrifugation, again decant the supernatant and filter it
by vacuum through a sintered glass filter.
7. Add 300 ml of Item 3 to 600 ml of serum obtained from Step 6.
Swirl gently to mix.
8. Dispense 2.14 cc in a vial that will hold a 2 ml fill and lyophilize.
~ials are reconstituted with 2.0 ml deionized distilled water. This serum is
used for a hypothyroid control serum in conjunction with all thyroid function
tests.
The fill solution after reconstitution will contain about 7 percent protein.
The resulting serum control should have a T3 uptake level less than 25% by the
Triosorb Method and a T4 concentration less than 5~31ug% by the Tetrasorb
Method. The serum used in this example may be obtained, for example, from
pregnant women or women on estrogen therapy.
A series of four separate experiments were carried out to show the
effect on sera when treated as to Examples 1 and 2. The resultant sera were
assayed by following the well recognized Triosorb and Tetrasorb protocols.
The results of these experiments (Table I) show that serum treated according
to the methods of this invention, may be used as a hypothyroid control serum
for tests being carried out in clinical laboratories.
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10;~ 85
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1039185
The results of experiment I show that the pooled normal serum was
euthyroid by both tests. After treatment the serum was hypothyroid for
both T3 uptake and T4 concentration.
The results for experiment II show that the hypothyroid values may
be altered upwardly when before treatment sera and after treatment sera are
intermixed, as in this experiment in a 2 to 1 ratio.
Experiment III was carried out with beef sera as representative of
animal sera which have been shown to have utility. As the results show,
pooled normal beef serum shows T3 uptake and T4 concentration in the euthyroid
range. After treatment, this sera behaves similar to human sera and is in the
hypothyroid range to T3 uptake and T4 concentration. As with human serum, the
T3 and T4 values may be increased by mixing various ratios of normal beef
serum and the prepared hypothyroid beef serum.
Experiment IV shows the expected hypothyroid T3 uptake and euthyroid
T4 concentration for pooled serum collected from pregnant women. After the
treatment outlined in Example 2, however, the serum becomes hypothyroid for
both T3 uptake and T4 concentration.
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