U.S. patent number 3,716,632 [Application Number 05/032,396] was granted by the patent office on 1973-02-13 for test composition and method for the in vitro determination of thyroid function.
This patent grant is currently assigned to Miles Laboratories, Inc.. Invention is credited to James Edward Christner, Marshall Lloyd Fader.
United States Patent |
3,716,632 |
Fader , et al. |
February 13, 1973 |
TEST COMPOSITION AND METHOD FOR THE IN VITRO DETERMINATION OF
THYROID FUNCTION
Abstract
The thyroid hormone content of blood serum can be readily
determined by using an in vitro radioactive thyroid function test
system containing a "protein error" pH indicator such as bromcresol
purple. Such a "protein error" indicator provides color and detects
the presence of the test sample, inhibits the formation of free
iodide and decreases the adsorption of radioactive tagged thyroid
substance to the walls of the container enclosing same.
Inventors: |
Fader; Marshall Lloyd (Elkhart,
IN), Christner; James Edward (Elkhart, IN) |
Assignee: |
Miles Laboratories, Inc.
(Elkhart, IL)
|
Family
ID: |
21864745 |
Appl.
No.: |
05/032,396 |
Filed: |
April 27, 1970 |
Current U.S.
Class: |
436/500; 252/645;
436/545; 436/804; 436/826 |
Current CPC
Class: |
G01N
33/78 (20130101); Y10S 436/804 (20130101); Y10S
436/826 (20130101) |
Current International
Class: |
G01N
33/487 (20060101); G01N 33/74 (20060101); G01N
33/78 (20060101); A61k 027/04 () |
Field of
Search: |
;424/1,2,7 ;252/301.1
;23/23R,23B |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Padgett; Benjamin R.
Claims
What is claimed is:
1. In a test composition for determining thyroid function
comprising a radioactive thyroid hormone and a buffer for
establishing the pH of the composition in a predetermined range,
the improvement which comprises adding to the composition a color
responsive amount of a protein error type pH indicator which
changes color in response to protein at the pH of the said
buffer.
2. A test composition as in claim 1 wherein the buffer is used to
maintain the pH of the composition at about 5.
3. A test composition as in claim 1 wherein the indicator is
selected from the group consisting of bromcresol purple,
chlorphenol red, tetrabromphenol blue, bromphenol blue, bromcresol
green and bromthymol blue.
4. A test composition as in claim 1 wherein the indicator is
bromcresol purple.
5. A test composition as in claim 1 wherein the indicator is
bromthymol blue.
6. A test composition as in claim 1 wherein the indicator is
present in a concentration of about 60 micrograms per milliliter of
composition.
7. A test composition as in claim 1 wherein the thyroid substance
is triiodothyronine.
8. In a method for determining thyroid function wherein blood serum
is added to a composition comprising a radioactive tagged thyroid
hormone and a buffer for establishing the pH of the composition in
a predetermined range, the improvement which comprises the addition
of a color responsive amount of a protein error type pH indicator
which changes color in response to protein at the pH of the buffer
prior to the addition of the serum.
9. A method as in claim 8 wherein the indicator is selected from
the group consisting of bromcresol purple, chlorphenol red,
tetrabromphenol blue, bromphenol blue, bromcresol green and
bromthymol blue.
10. A method as in claim 8 wherein the indicator is bromcresol
purple.
11. A method as in claim 8 wherein the indicator is present in a
concentration of about 60 micrograms per milliliter of
composition.
12. A method as in claim 8 wherein the thyroid substance is
triiodothyronine.
13. A method of inhibiting the adsorption of radioactive
triiodothyronine from an aqueous solution thereof on the walls of a
polyethylene container which comprises adding to said aqueous
solution an adsorption inhibiting amount of a protein error type pH
indicator.
14. A method as in claim 13 wherein the indicator is selected from
the group consisting of bromcresol purple, chlorphenol red,
tetrabromphenol blue, bromphenol blue, bromcresol green and
bromthymol blue.
15. A method as in claim 13 wherein the indicator is bromthymol
blue.
16. A method as in claim 13 wherein the container is prewashed with
an aqueous solution of bromcresol purple at a concentration of from
50 to 70 micrograms per milliliter of water.
Description
BACKGROUND OF THE INVENTION
Numerous tests have recently been developed to determine thyroid
function in vitro without exposure of the patient to ionizing
radiation. For example, specific plasma proteins known as
thyroxine-binding proteins or TBP are the primary binding sites for
the hormones produced by the thyroid gland. Since the TBP have a
strong affinity for thyroid hormone, they will bind any free
thyroid hormone available until they become saturated. In this
test, such hormone is made available to the TBP in a sample of the
patient's serum and a secondary binding site consisting of a resin
powder or cross-linked dextran (Sephadex) is provided to take up
any of the added radioactive triiodothyronine or thyroxine
(hereinafter referred to as T.sub.3 or T.sub.4, respectively) not
bound by the TBP. Thus, the test provides an estimation of the
saturation level of the TBP in a given serum sample which is an
indirect but reliable indication of thyroid function. A large resin
uptake indicates hyperthyroidism whereas a small resin uptake
indicates hypothyroidism.
Two such tests for thyroid function are described in U.S. Pat. Nos.
3,206,602 and 3,451,777. Since no color change is involved in
either test, the operator can become confused if the procedural
steps are interrupted. In addition, the decomposition of
radioactive iodinated T.sub.3 to the free iodide form, combined
with considerable adsorption of the aqueous buffered radioactive
T.sub.3 solution to glass or plastic containers, produces a
significant error in the determination of the thyroid hormone
content of the serum sample.
SUMMARY OF THE INVENTION
The main object of the present invention is to provide a thyroid
function test solution containing a pH indicator which possesses
the three fold advantage of (1) providing a color change when the
serum sample is added thereto (2) inhibiting the formation of free
iodide from radioactive iodinated T.sub.3 and (3) decreasing the
adsorption of radioactive iodinated T.sub.3 to the walls of the
vessel containing the same.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Although thyroid function tests may be performed in many different
ways, one common method involves adding an appropriate amount of a
buffered aqueous solution of radioactive triiodothyronine or
thyroxine and serum to a column of ion exchange resin such as
Amberlite IRA 400 made by Rohm and Haas Co. of Philadelphia, Pa. or
a cross linked dextran such as Sephadex G-25 made by Pharmacia
Corp. of Uppsala, Sweden. The column is then drained and the
radioactivity measured in a gamma ray counting instrument for 1
minute. The column is thereafter eluted with distilled water and
the radioactivity remaining on the column is again measured for 1
minute. The percent retention is then calculated by comparing the
two readings. Retentions of 41 percent to 61 percent are considered
normal with cross-linked dextran (Sephadex) columns and 25 percent
to 35 percent with resin columns.
It has now been found that by adding a "protein error" pH indicator
to an aqueous radioactive T.sub.3 solution prior to combining with
the serum sample or by adding same to the combined T.sub.3 solution
and serum sample, one can achieve a highly improved test
system.
The pH indicator employed in the present invention is of the
protein error type which changes color in the presence of albumin
without a significant actual change in pH. When such an indicator
is added in a concentration of about 5. to 70 micrograms per
milliliter of aqueous I.sup.125 -T.sub.3 reagent buffered at a pH
of 5 the reagent changes from its initial color to a second color
when the serum is added. This second color acts as a marker which
will appear at varying heights on the resin or dextran column
depending on the last operation step completed in the test
procedure, since the color will move with the solvent front.
A preferred indicator is bromcresol purple which changes color from
yellow to purple within the pH range of 5.2 to 6.8. When mixed with
aqueous radioactive T.sub.3 solution, the yellow-orange color will
turn to a grey-green color when serum is added at a ratio of 0.05
milliliter of serum to 0.45 milliliter of reagent mixture. Thus,
the green color indicates the presence of the serum. Other
satisfactory indicators which can be employed depending on the pH
of the buffer system are (1) chlorphenol red, which changes from
red to violet at a pH of 6.5 (2) tetrabromphenol blue, which
changes from yellow to blue at a pH of 3.0 (3)bromphenol blue,
which changes from yellow to purple at a pH of 3.0 (4) bromcresol
green, which changes color from yellow to blue at a pH of 3.0 and
(5) bromthymol blue, which changes from green to yellow at a pH of
6.5. In practice, the colors of the color change are not important
as long as a difference can be easily seen.
Preferably, the improved T.sub.3 test reagent with pH indicator of
this invention will contain the following:
Ingredient Amount
__________________________________________________________________________
Anhydrous citric acid 43.2 grams Dibasic sodium phosphate 125.0
grams Formaldehyde (38% aqueous solution) 8.1 milliliters I.sup.125
triiodothyronine 4.0 micrograms Triiodothyronine unlabelled 26.0
micrograms Bromcresol purple 0.18 grams Distilled water q.s. to 3.0
liters
__________________________________________________________________________
In addition to the color change which occurs in the use of a pH
indicator of the type disclosed, another advantage resides in the
unforeseen property of the indicator to inhibit the formation of
free iodide from the I.sup.125 -T.sub.3 employed in the T.sub.3
reagent. This is important, since the gamma counters cannot
distinguish between I.sup.125 -T.sub.3 and free I.sup.125, and all
of the dose I.sup.125 is counted initially during the test
procedure. Washing with water removes any free I.sup.125 from the
column, since only the hormonal materials remain thereon. Thus, if
the percent of free iodide is above 5 percent of the total dose
counts, the T.sub.3 ratio is subject to a significant error of from
5 percent to 10 percent.
In a series of tests, a buffered aqueous solution of I.sup.125
-T.sub.3 containing 60 micrograms of bromcresol purple per
milliliter prepared as shown in the foregoing example was stored
under various conditions in a polyethylene container and the
percent of free iodide formed was compared with a similar solution
containing no pH indicator. The initial solutions employed
contained about 2 percent free iodide. After 90 days at room
temperature, the radioactive T.sub.3 solution containing the
indicator showed 2.25 percent free iodide compared to 5.2 percent
free iodide in the control sample containing no indicator. After 60
days at 40.degree. C., the T.sub.3 test solution with pH indicator
contained 4.25 percent free iodide, whereas the control sample had
a free iodide content of 9.0 percent.
A still further advantage of the use of a pH indicator in T.sub.3
test solution is to inhibit the adsorption of radioactive T.sub.3
to the walls of a plastic or glass container. Propylene glycol and
albumin have been used in the past to reduce such adsorption but
these additives interfere with the thyroid function test. In actual
tests, aqueous radioactive T.sub.3 solutions containing one of
several indicators at a concentration of 60 micrograms per
milliliter of solution were rotated on a wheel in a polyethylene
container for 14 days and the percent activity remaining was
compared with a control sample containing no indicator. The results
obtained showed that over 90 percent of the radioactivity (after
correction for physical decay) remained in those solutions
containing bromthymol blue, chlorphenol red, bromcresol purple and
tetrabromphenol blue, whereas with solutions containing phenol red
or bromcresol green, 80 percent of the radioactivity remained,
while those containing bromphenol blue retained 70 percent of the
radioactivity in solution. Under the same test conditions, the
control sample not containing one of the mentioned indicators
retained only 32% of the radioactivity in solution. Similar results
are obtained with glass containers.
Decreased adsorption of like magnitude has been observed when
plastic or glass containers are prewashed prior to filling with
radioactive -T.sub.3 reagent devoid of indicator. The term
"prewashed" means contacting (such as by rinsing, soaking or
flushing) the interior surfaces of said containers with an aqueous,
buffered solution of one of the pH indicators hereinbefore
disclosed. Bromcresol purple or bromthymol blue are the preferred
indicators. These are employed at a concentration of from 50 to 70
micrograms per milliliter of water buffered to a pH of about 5.
It is to be understood that the T.sub.3 employed in the test
compositions of this invention can be made radioactive with either
I.sup.125 or I.sup.131, although I.sup.125 is preferred, since it
has a half-life of 56 days compared to I.sup.131 which has a
half-life of 8 days.
* * * * *