NO751877L - - Google Patents

Description

"Procedure for serum examination

of hypothyroidism"

Circulating Tg and T^ are bound to many constituents in the blood of which the thyroxine-binding globulin (TBG) fraction contains the main binding sites in a certain number. The binding of Tg and to the TBG molecule is a competitive protein binding, i.e. unbound will replace Tg and T4 which are already present in the molecule.

Since there is only 1-2 mg of TBG/100 ml of normal serum, the binding positions, which are equivalent to approx. 20 micrograms

per 100 ml of normal serum, easily by a small increase of the T^ concentration.

in'

In hyperthyroidism, the binding positions in the TBG molecule are almost saturated with Tg and T^. In hypothyroidism, the binding positions are highly unsaturated, which results in an increased ability of the TBG molecule in serum to absorb Tg.

The Triosorb determination and other similar tests on Tg absorption work according to the principle of competitive protein binding. Serum-Tg and -T^ are primarily bound to the binding positions in the TBG molecule. The number of unsupported binding positions is determined by adding radioactive Tg (Tg+) to serum in the presence of an adsorbent. When Tg+ is added to serum, any excess that is not bound to the binding sites in the TBG molecule in the serum will be adsorbed to the adsorbent. Resin sponges are the adsorbent for the Triosorb test. In hyperthyroidism, for example, most of the TBG binding positions are occupied by Tg and T4, and thus the added Tg+ will not be occupied by the endogenous TBG molecules, but will be taken up by the test adsorbent. In hypothyroidism, the reverse is the case. Thus, the amount of radioactive Tg that is bound by the adsorbent will directly indicate the patient's thyroid condition.

The tetrasorb test on T4 is also based on the principle of competitive protein binding. To determine the T^ concentration, T4 is extracted from the serum and freed from its binding protein, TBG. The serum extract is then added to a solution containing a limited amount of exogenous TBG to which radioactive T4 (T4+) is bound. A displacement reaction takes place in which

T^ in the extract displaces T4+ from the exogenous TBG. This displaced T^+ is then adsorbed on the resin sponge which is used as

adsorbent. When more T4 is added, more T^+ is displaced from TBG. The amount of T4+ displaced from 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 there are normal amounts of TBG in the serum, these types of thyroid function tests demonstrate the relevant condition in the patient. When TBG and T4 levels are elevated, such as in pregnancy or as a result of oestrogenic introduction in the form of oral contraceptives, Tg uptake tests indicate that the patient is hypothyroid. However, tests that measure total T4 indicate a euthyroid or sometimes a hyperthyroid state. In a simple way, it can be said that pregnancy or estrogen addition results in an increase in the number of TBG molecules causing an increase in hormone binding positions with a concomitant increase in thyroid hormone levels. Thyroid function tests performed on these sera at this time show an increased Tg uptake, indicating a hypothyroid function and an increased T4~ level, indicating a hyperthyroid function. It is the purpose of this invention to describe a control serum prepared from these types of sera and normal serum which will serve as a hypothyroid control serum for T4 tests as well as Tg uptake tests. By normal serum, the applicants mean serum with normal Tg and T4 values and which can be human, ox, sheep, goat or other acid serum. Only horse serum has proven to be unusable.

A serum is hyperthyroid according to the tetrasorb and triosorb tests if the percentage Tg absorption is greater than 35, and the T4 concentration is over 14.5 micrograms per 100 ml of serum. A serum is euthyroid if the percentage Tg uptake is 25 to 35 and the T4 concentration is 5.3-14.5 mkg per 100 ml of serum. Hypothyroid serum has a Tg uptake of less than 25 percent and less than 5 mljg T4

per 100 ml of serum.

Removal of Tg and T^ from normal serum or serum containing elevated TBG levels, which according to the methods indicated below, results in a serum judged to be hypothyroid both by methods that measure T^ concentration and Tg uptake.

Example 1

Method

1. Article 2 is added to 2400 ml of article 1 in a 4 litre

Erlenmeyer flask (approx. 20% charcoal to volume of serum) .

2. It is shaken gently at room temperature until the coal particles are dispersed in the liquid. 3. The mixture is placed at 4°C. It is stirred gently for approx.

24 hours.

4. At the end of 24 hours, centrifuge the mixture at high speed in a refrigerated centrifuge (34,800 x g). 5. The supernatant liquid is decanted and it is centrifuged again as in step 4. 6. After the second centrifugation, the supernatant liquid must again be decanted and then filtered by vacuum through a millipore filter. 7. The filtrate (1200 ml) is poured into a suitable shard of glass suitable for lyophilization and lyophilized. 8. The lyophilized material is transferred to a two liter Erienmeyer flask and article 3 is added. 9. Let the mixture stand for 30 minutes and the dissolution process is then assisted by gentle shaking. 10. 300 ml of article 1 is then added to the serum mixture obtained

in step 9.

11.2.14 ml is transferred to a medicine glass that can contain 2 ml and then lyophilized.

Medicine glasses are reconstituted with 2.0 ml of deionized, distilled water. This serum is used as a hypothyroid control in connection with all thyroid function tests, i.e. the resulting serum control must have a Tg uptake level of less than 25% according to the Triosorb method and a T^ concentration of less than 5.3 micrograms/ml according to the Tetrasorb method .

Modifications are of course possible in Example 1. Centrifugation (steps 4 and 5) can be eliminated. The Millipore filter (step 6) can of course be an Ertel apparatus. The serum can be human or animal serum. The requirement to transfer the mixture (step 11) can of course be modified or avoided altogether. The mixing time (step 3) can be shortened or extended within the range of 3-30 hours. The percentage of carbon that is added can vary from 5-20 per cent, with an area of from approx. 10 to approx. 20 percent is preferred.

This method removes over 99% of Tg and T^ from the starting serum, and a Tg- and T^-free serum is effectively produced, while the total protein concentration, pH or T^-binding capacity of the serum is not significantly affected.

The following example illustrates the method used to remove Tg and T4 from serum containing elevated TBG levels. Modifications in this example similar to those indicated for

example 1 are possible and are covered by the invention.

Example 2

Method

1. Article 2 is added to article 1 in a 2 liter Erlenmeyer flask (approx. 20% carbon weight to volume of serum). 2. It is shaken carefully at room temperature for one minute so that the carbon particles are dispersed in the liquid. 3. The mixture is placed at 4°C and stirred very carefully for approx. 24 hours. 4. After 24 hours, the mixture is centrifuged at high speed in a cooled centrifuge (34,800 x g).

5. The remaining liquid is decanted and it is centrifuged again as

1 step 4.

6. After another centrifugation, the remaining liquid is decanted again and filtered in vacuum through a sintered glass filter. 7. Add 300 ml of Article 3 to 600 ml of serum obtained from step 6. Shake gently to mix the ingredients.

8. 2.14 ml is transferred to a medicine glass that can contain

2 ml and it is lyophilized.

The medicine glasses are reconstituted with 2.0 ml of deionized, distilled water. This serum is used as a hypothyroid control serum in connection with all thyroid function tests. After reconstitution, the filling solution will contain approx. 7 percent protein. The resulting serum control should have a Tg uptake level of less than 25% by the Triosorb method and a T^ concentration of less than 5.3 µg by the Tetrasorb method. Serum used in this example can, for example, be obtained from pregnant women or from women on estrogen therapy.

A series of four separate experiments were performed to show the effect on sera when treated as in Examples 1 and 2.

The resulting sera were tested following the well known Triosorb and Tetrasorb protocols. The results of these tests (table 1) show that serum treated by the method according to the invention can be used as a hypothyroid control serum for tests carried out in clinical laboratories.

The results from experiment I show that the total normal serum was euthyroid according to both tests. After treatment, the serum was hypothyroid for both Tg uptake and T^ concentration.

The results from trial II show that the hypothyroid values

can change upwards when sera from before the treatment and sera after the treatment are mixed together, as in this experiment in a ratio of

2 to 1.

Experiment III was performed with bull sera as representatives

for animal sera that have proven useful. As the results show, pooled normal bovine serum shows Tg uptake and T^ concentration in the euthyroid range. After treatment, these sera behave on

the same way as human sera and is in the hypothyroid range for both Tg uptake and T^ concentration. As with human serum, the Tg and T4 values can be increased by mixing different ratios of normal bovine serum and the prepared hypothyroid bovine serum.

Trial IV shows the expected hypothyroid Tg uptake and euthyroid T4 concentration for pooled serum obtained from pregnant women. After the treatment indicated in example 2, however, the serum becomes hypothyroid for both Tg uptake and T4 concentration.

Claims (11)

1. Procedure for achieving a hypothyroid serum control, characterized by the following steps: A. addition of neutral decolorizing charcoal to blood serum in an amount of approx. 5-20 percent based on weight of coal to volume of serum, B. mixing of the above mixture for approx. 24 hours at a temperature of approx. 4°C, C. centrifugation of the resulting slurry twice at 34,800 x g and at a temperature of approx. 4°C, D. filtering, of the resulting supernatant liquid and E. lyophilization of the thus obtained supernatant liquid. 2. Method according to claim 1, characterized by the addition of untreated normal serum in an amount of approx. 10 to approx. 35 percent of the serum volume in step A to the filtered supernatant in step D. 3. Method according to claim 1, characterized in that animal blood serum is used as serum. 4. Method according to claim 1, characterized in that human blood serum is used as serum. 5. Method according to claim 1, characterized in that ox blood serum is used as serum. 6. Method according to claim 4, characterized in that serum is used which is T^-euthryoid, -hypothyroid or -hyperthyroid and Tg-euthyroid or -hypothyroid. 7. Method according to claim 6, characterized by the use of serum that is Tg-hypothyroid. 8. Method according to claim 6, characterized by the use of serum that is T4-euthyroid and Tg-euthyroid. 9. Control serum judged to be hypothyroid according to methods that measure T^ concentration and Tg uptake, characterized in that it includes blood serum which has have been processed by the method according to claim 1. 10. Clinical method for measuring a person's serum triiodothyronine uptake level and thyroxine concentration, characterized in that it comprises the use of a single control serum standard as a hypothyroid reference control serum for both triiodothyronine uptake and thyroxine concentration tests. 11. Method according to claim 10, • characterized in that the preparation of the reference control serum comprises the following steps A. addition of neutral decolorizing charcoal to blood serum in an amount of approx. 5-20 percent based on weight of carbon to volume of serum, B. mixing of the above mixture at a temperature of approx. 4°C, C. centrifugation of the resulting slurry twice at 34,800 x g and at a temperature of approx. 4°Cf D. filtering the resulting supernatant and E. lyophilization of the thus obtained supernatant liquid.