METHOD AND STANDARD SOLUTION FOR THE DETERMINATION OF THYROXINE (T4) OR TRIIODOTHYRONINE (T3)

METHODE ET SOLUTION ETALON POUR LE TITRAGE DE LA THYROXINE (T4) OU DE LA TRIIODOTHYRONINE (T3)

English Abstract

A b s t r a c t

A standard solution containing bovine thyroxine-binding
globulin (TBG) and thyroxine or triiodothyronine
dissolved in a buffer solution is used for calibration
in a method for the determination of thyroxine (T4) or
triiodothyronine (T3) in serum.

Claims

The embodiments of the invention in which an exclusive
property or privilege is claimed are defined as
follows:
1. A method for the determination of thyroxine
(T4) or triiodothyronine (T3) in serum whereby a
standard solution is used for the calibration which
contains thyroxine-binding globulin (TBG) and
thyroxine or triiodothyronine dissolved in a buffer
solution, wherein said TBG is bovine TBG.
2. A method as claimed in claim 1, wherein a
solution is used as a further standard solution which
contains bovine TBG dissolved in a buffer solution but
contains no thyroxine or triiodothyronine.
3. A method as claimed in claim 1 or 2, wherein
said standard solution further contains albumin.
4. A method as claimed in claim 3, wherein said
albumin is bovine serum albumin.
5. A method as claimed in claim 3, wherein said
standard solution contains 40 to 80 mg/ml of said
albumin.
6. A method as claimed in claim 4, wherein said
standard solution contains 40 to 80 mg/ml of said
albumin.
7. A method as claimed in claim 3, wherein said
standard solution contains 50 to 70 mg/ml of said
albumin.
8. A method as claimed in claim 4, wherein said
standard solution contains 50 to 70 mg/ml of said
albumin.


9. A method as claimed in claim 1, 2, 4, 5, 6,
7 or 8, wherein said standard solution contains 5 to
30 µg/ml of said bovine TBG.
10. A method as claimed in claim 1, 2, 4, 5, 6,
7 or 8, wherein said standard solution contains 9 to
20 µg/ml of said bovine TBG.
11. A method as claimed in claim 1, 4, 5, 6, 7
or 8, wherein said standard solution contains 5 to 500
ng/ml of thyroxine.
12. A method as claimed in claim 9, wherein said
standard solution contains 5 to 500 ng/ml of
thyroxine.
13. A method as claimed in claim 10, wherein
said standard solution contains 5 to 500 ng/ml of
thyroxine.
14. A method as claimed in claim 1, 4, 5, 6, 7
or 8, wherein said standard solution contains 5 to 300
ng/ml of thyroxine.
15. A method as claimed in claim 9, wherein said
standard solution contains 5 to 300 ng/ml of
thyroxine.
16. A method as claimed in claim 10, wherein
said standard solution contains 5 to 300 ng/ml of
thyroxine.
17. A method as claimed in claim 1, 4, 5, 6, 7
or 8, wherein said standard solution contains 0.5 to
12 ng/ml of triiodothyronine.
18. A method as claimed in claim 9, wherein said
standard solution contains 0.5 to 12 ng/ml of
triiodothyronine.


19. A method as claimed in claim 10, wherein
said standard solution contains 0.5 to 12 ng/ml of
triiodothyronine.
20. A method as claimed in claim 1, 4, 5, 6, 7
or 8, wherein said standard solution contains 0.5 to 8
ng/ml of triiodothyronine.
21. A method as claimed in claim 9, wherein said
standard solution contains 0.5 to 8 ng/ml of
triiodothyronine.
22. A method as claimed in claim 10, wherein
said standard solution contains 0.5 to 8 ng/ml of
triiodothyronine.
23. A method as claimed in claim 1, 2, 4, 5, 6,
7, 8, 12, 13, 15, 16, 18, 19, 21 or 22, wherein said
buffer solution has a pH range from 6 to 8.
24. A method as claimed in claim 1, 2, 4, 5, 6,
7, 8, 12, 13, 15, 16, 18, 19, 21 or 22, wherein said
buffer solution contains a GOOD buffer.
25. A method as claimed in claim 1, 2, 4, 5, 6,
7, 8, 12, 13, 15, 16, 18, 19, 21 or 22, wherein said
buffer solution contains 30 to 150 mmol/l buffer.
26. In a method for the determination of
thyroxine (T4) or triiodothyronine (T3) in serum in
which a calibration is carried out with a standard
solution containing thyroxine-binding globulin (TBG)
and thyroxine or triiodothyronine in a buffer
solution, the improvement wherein said TBG is bovine
TBG.
27. A standard solution for the determination of
thyroxine or triiodothyronine containing bovine TBG


and thyroxine or triiodothyronine dissolved in a
buffer solution.
28. A standard solution as claimed in claim 27,
wherein it contains 5 to 30 µg/ml bovine TBG and 30 to
150 mmol/l buffer.
29. A standard solution as claimed in claim 27
or 28, further containing albumin.
30. A standard solution as claimed in claim 29,
wherein said albumin is present in an amount of 40 to
80 mg/ml.
31. A standard solution as claimed in claim 27,
28 or 30, which contains 5 to 500 ng/ml thyroxine.
32. A standard solution as claimed in claim 29,
which contains 5 to 500 ng/ml thyroxine.
33. A standard solution as claimed in claim 27,
28 or 30, which contains 5 to 300 mg/ml thyroxine.
34. A standard solution as claimed in claim 27,
28 or 30, which contains 0.5 to 12 ng/ml
triiodothyronine.
35. A standard solution as claimed in claim 29,
which contains 0.5 to 12 ng/ml triiodothyronine.
36. A standard solution as claimed in claim 27,
28 or 30, which contains 0.5 to 8 ng/ml
triiodothyronine.
37. A standard solution as claimed in claim 29,
which contains 0.5 to 8 ng/ml triiodothyronine.

Description

- 210364~


D e s c r i p t i o n

The invention concerns a method for the determination of
thyroxine (T4) or triiodothyronine (T3) in serum using a
standard solution as well as this standard solution.

The thyroid hormones are present in blood mainly in a
protein-bound form. The most important carrier protein
for this is thyroxine-binding globulin, which is denoted
TBG, to which ca. 80 % of the total thyroxine and
triiodothyronine present in blood is bound.
Approximately 5 to 10 ~ of the total thyroxine is
present bou~d to albumin and about 10 to 15 % of the
total thyroxine is present bound to pre-albumin. The
totality of these proteins is ~lso referred to as
thyroxine-binding protein (TBP). Only very small
proportions of thyroxine and triiodothyronineare present
in blood in a free for~; usually ca. 0.03 % of T4 or
0.3 % of T3. Only the released hormones are
physiologically active whereas the bound hormones T3 and
T4 circulate in the blood as a metabolically inert
transport form and serve as a buffer to regulate the
level. It is therefore important to determine the total
thyroxine content as well as the proportion of free
thyroxine and free triiodothyronine in blood since this
represents the functional thyroid status independent of
changes in the concentration or saturation of the
thyroxine binding protein.

Various methods are known for the determination of total
thyroxine (T4), free thyroxine (FT4) and
triiodothyronine (T3). A difficulty in all of these
known methods is the provision of a standard which is
suitable for the calibration.

2 ~

For the calibration it is necessary to produce human
serum standards with particular thyroxine or
triiodothyronine concentrations and a defined FT4 or FT3
content. Human serum has previously been used as
starting material for this. Firstly all the thyroxine
and triiodothyronine in a free as well as in a bound
form has to be removed from the human serum and then the
human serum treated in this way has to again be
supplemented with defined amounts of thyroxine or
triiodothyronine. Various methods are described in the
literature for this purpose such as treatment with
active charcoal, with ion exchangers or with carrier-
bound anti-T4 or anti-T3 antibodies. These methods are
all very complicated. A further problem is that each
human serum has a different composition thus leading to
large variations in the individual lots and as a
consequence the reproducibility leaves much to be
desired.

A further disadvantage of these known standard solutions
for the determination of T3 and T4 is that these
solutions, which have the composition of human serum,
cannot be stored for longer periods since on the one
hand the protein which is present is gradually denatured
and on the other hand there is a shift in the set
equilibrium between bound and free T3 and T4.

It is known from EP-A O 337 467 that in order to solve
the above-mentioned problems a standard solution can be
used for a method to determine thyroxine or
triiodothyronine which is still stable after long
storage and can be produced in a simple manner using
relatively cheap startin~ materials by using at least
one standard solution for the calibration that contains

~03~'16

TBG and thyroxine or triiodothyronine dissolved in a
buffer solution.

It has now been found that a further quite considerable
improvement of the stability on storage can be achieved
if TBG from bovine serum is used in the aforementioned
methods.

The invention therefore concerns a method for the
determination of thyroxine (T4) or triiodothyronine (T3)
in serum in which a standard solution is used for the
calibration which contains thyroxine-binding globulin
(TBG) and thyroxine or triiodothyronine dissolved in a
buffer solution which is characterized in that bovine
TBG is used.

Whereas when using the known methods the standard
solution in liquid reagent form can only just achieve a
minimum storage capability of 8 weeks and therefore has
to be stored in a lyophilized form, the standard
solution used according to the present invention is
stable on storage in a liquid form for more than 10
times as long. According to the results obtained thus
far the storage stability in a liquid form will attain
at least 18 months and after this time period there is
still a full binding capacity for T4 and T3 whereas the
corresponding standard solution with human TBG even when
stored below 10C already yields erroneous values which
are 20 % too high after 12 weeks. Moreover the bovine
serum used to isolate bovine TBG is much more readily
obtainable than human serum.

The superior stability of the standard solution
according to the present invention can be seen in

210~

Table 1. From this it is apparent that with human TBG
(H-TBG) the analyte stability decreases rapidly so that
erroneously high values are found which already after 3
months are 20 % higher (i.e. 120 ~ of the initial value)
than with a fresh solution. In contrast using the
standard solution according to the present invention no
change whatsoever can be observed even after 12 months
i.e. the analytical values are unchanged and correct at
100 %. The stated contents in percent relate to the
measured recovery of analyte compared to a fresh
product. Here it should be noted that analyte bound to
TBG, i.e. T4 or T3, is not detected at all and an
increase in the value for T4 or T3 means that the
binding to TBG has deteriorated thus yielding false
values compared to fresh natural TBG. The limit which is
still ~ust acceptable is a change of + 5 %.

Various methods are known for determining thyroxine and
triiodothyronine. Methods of determination are for
example described in Nuc. Compact 16 (1985), 321-327, J.
Clin. Immunoassay 7 (1984), 192-205, and J. Clin. Chem.
~lin. Biochem. 22 (1984), 895-904. Determination methods
based on immunoassays are used especially for
determining thyroid hormones.

In a known method fsr the determination of thyroxine or
triiodothyronine in serum based on an immunoassay
technique labelled T4 or T3 and T4 or T3 from the sample
usually compete for a binding partner such as for
example an anti-T4 antibody. By adjusting the reaction
conditions such as the concentration of the individual
components, incubation period etc., it is possible to
either determine the total thyroxine or total
triiodothyronine or the free thyroxine or free
triiodothyronine. For this the amount of labelled T3 or

2~0364~
-- 5 --

T4 bound to the anti-T4 or anti-T3 antibody or non-bound
labelled T3 or T4 is determined via known detection
reactions. After measuring the label the content of T3
or T4 can then be calculated by comparing the value
obtained to values obtained with known T3 or T4
concentrations. For this it is necessary to establish a
calibration curve in which the measurement signal (e.g.
the absorbance value) is plotted against the T3 or T4
concentration. In the case of thyroxine it is expedient
to establish a calibration curve from six thyroxine
solutions with different concentrations since there is
no linear relationship which would allow a two-point
standard measurement.

The method according to the present invention is
suitable for the determination of free thyroxine (FT4 ),
free triiodothyronine (FT3) as well as of total
thyroxine (T4) and thyroxine (T3) in serum.

The thyroxine-binding bovine globulin can be isolated
from bovine sera in a known manner. The standard
solution used according to the present invention
preferably contains 5 to 30 ~g/ml bovine TBG. It is
particularly preferred to use bovine TBG in a
physiological amount which is in the range from 9 to
20 ~g/ml TBG which for example can be gathered from the
literature references Lab. Med. 6 (1982), 27-2~, and J.
Clin. Chem. Clin. Biochem. 23 (1985) 117-127.

If the standard solution is to be used to determine
thyroxine it additionally contains thyroxine. The
thyroxine is preferably present in an amount of 5 to
500 ng/ml. It is particularly preferable to add a
physiological amount of thyroxine to the standard

~1~3~


solution i.e. an amount in the range from 5 to
300 ng/ml.

If the standard solution is to be used to determine
triiodothyronine it additionally contains
triiodothyronine. The triiodothyronine is preferably
used in an amount of 0.5 to 12 ng/ml. It is particularly
preferable to add triiodothyronine in a physiological
amount i.e. in the range of 0.5 to 8 ng/ml.

The standard solution used for the method according to
the present invention preferably also contains albumin
which improves the stability of the solution in addition
to bovine TBG and thyroxine or triiodothyronine in
buffer solution. In this connection it is not necessary
to add human serum albumin, which is of course suitable,
to the standard solution preferred according to the
present invention. Equally suitable are the universally
and easily obtainable and much more preferable albumins,
for example from bovine or horse serum. Human serum
albumin, bovine serum albumin or horse serum albumin and
in particular bovine serum albumin is preferably used as
albumin. The albumin is preferably used in an amount of
40 to 80 mg/ml solution. It is particularly preferable
to use albumin in a physiological amount which is in a
range from 50 to 70 mg/ml.

In order to produce the standard solution, bovine TBG
and, if desired, albumin are dissolved in a buffer
solution. All buffers which have a pH range of 6.0 to
8.0 are suitable as the buffer system, preferably those
with a pH value of 6.5 to 7.5. GOOD buffers are
preferably used, for example HEPES or TRIS buffer. The
buffer concentration is not critical, the buffer is
preferably used at a concentration of 30 to 150 mmolJl.

6~

-- 7

The standard solution particularly preferably contains
50 to 100 mmol/l buffer.

Subsequently the thyroxine or triiodothyronine in
appropriate concentrations is added to this solution. In
this process an equilibrium is established between
protein bound and free thyroid hormones which i5 similar
to the equilibrium which is present in vivo in blood.
This solution is also stable over long periods of
storage because of its composition. Since it is produced
from standardized individual substances it always has a
uniform composition and therefore yields reproducible
values~

In order to establish a calibration curve, several
standard solutions - as a rule four to six - are used
with different contents of thyroid hormones so that a
curve can be drawn from the values obtained. In a
preferred embodiment of the method according to the
present invention a solution is used as the first
standard solution which only contains bovine TBG and, if
desired, albumin in a buffer solution but contains no
thyroid hormone. Solutions with defined hormone contents
are then used for the further calibration.

The invention also concerns a standard solution for the
determination of thyroxine or triiodothyronine which
contains bovine TBG and thyroxine or triiodothyronine
dissolved in a buffer system.

The standard solution preferably contains 5 to 30 ~g/ml
bovine TBG. It is particularly preferred that a
physiological amount Df bovine TBG, i.e. 9 to 20 ~g/ml,
is present in the standard solution.

36~
-- 8

In a preferred embodiment the standard solution
additionally contains albumin for stabilization. In this
case the albumin is preferably present in an amount of
40 to 80 mg/ml. It is particularly preferable to use
physiological amounts of albumin i.e. in the range of 50
to 70 mg/ml albumin.

~he standard solution is produced by dissolving bovine
TBG and, if desired, albumin in a buffer system and
adding the amount of thyroxine or triiodothyronine
desired in each case. In this connection the amount of
thyroxine is preferably in the range of 5 to 500 ng/ml
and particularly preferably in the physiological range
of 5 to 300 ng/ml. If the standard solution is used to
determine triiodothyronine it then contains
triiodothyronine prefe~ably in an amount of 0.5 to
12 ng/ml and particularly preferably in a physiological
amount in the range of 0.5 to 8 ng/ml.

The buffer system preferably has a pH in the range 6.0
to 8.0 and is preferably a GOOD buffer.

According to the invention a standard solution with a
superior stability is provided which can be produced in
a simple manner from easily obtainable starting
materials.

~3~4~
_ 9 _

Example
Isolation of bovine TBG

Bovine plasma is brought to a concentration of 2 mol/l
ammonium sulfate, the supernatant is decanted off
diluted to 1 mol/l ammonium sulfate and applied to a
chromatography column (Sepharose to which T4 is bound).
It is washed in succession with:

1. 50 mmol/l Tris pH 7.5, 100 mmol/l sodium chloride
2. 50 mmol/l Tris pH 7.5, 750 mmol/l sodium chloride
3. 50 mmol/l Tris pH 7.5, 100 mmol/l sodium chloride
4. 50 mmol/l Tris pH 7.5, 100 mmol/l sodium chloride
5 mmol/l 8-anilino-naphthaline sulfonic acid.

The eluate is concentrated in an osmotic cell and 8-
anilino-naphthaline sulfonic acid is removed by
dialysing for two days against 5 mmol/1 sodium phosphate
pH 7.5, 10 mmol/l sodium chloride.

Example 2
Stability of T3/T4 calibrators

Composition of the calibrators:

6 % bovine serum albumin,
50 mmol/l Hepes buffer, pH 7.4
15 ~g/ml bovine TBG

FT3 content: 0, 2, 7, 15, 30 pgJml
or FT4 content: 1, 10, 20, 40, 80 pg/ml

~lQ3~

-- 10 --

The following Table shows the stability of T3/T4
calibrators.

Table 1:

Storage period Recovery1) with
at 4 - 8C an analyte content
(months) of 2 - 30 pg/ml
.
T3 without TBG 3 118
6 130
12

T3 with human TBG 3 115
6 130
12 138

T3 with bovine TBG 3 100
6 97
12 99

1) The recovery shows the measurement value found for T3
in relation to the measurement value for T3 at the
beginning of storage (100 ~).