U.S. patent number 3,659,104 [Application Number 05/051,005] was granted by the patent office on 1972-04-25 for method of measuring serum thyroxine.
This patent grant is currently assigned to Yissum Research Development Company of the Hebrew University of Jerusalem. Invention is credited to Amirav Gordon, Jack Gross, Lloyd Alan Schick.
United States Patent |
3,659,104 |
Gross , et al. |
April 25, 1972 |
**Please see images for:
( Certificate of Correction ) ** |
METHOD OF MEASURING SERUM THYROXINE
Abstract
A new and improved in vitro concept in measuring serum thyroxine
(T-4) is sclosed which employs an alkaline crosslinked dextran gel
(Sephadex) column to dissociate and separate the T-4 from the serum
protein in a single operation. An isotope dilution technique
combined with saturation analysis is used to estimate the T-4
content in serum.
Inventors: |
Gross; Jack (Jerusalem,
IL), Gordon; Amirav (Jerusalem, IL),
Schick; Lloyd Alan (Elkhart, IN) |
Assignee: |
Yissum Research Development Company
of the Hebrew University of Jerusalem (N/A)
|
Family
ID: |
21968790 |
Appl.
No.: |
05/051,005 |
Filed: |
June 29, 1970 |
Current U.S.
Class: |
436/500; 250/304;
436/529; 436/804; 436/826; 250/303; 436/545; 436/825 |
Current CPC
Class: |
G01N
33/78 (20130101); Y10S 436/804 (20130101); Y10S
436/826 (20130101); Y10S 436/825 (20130101) |
Current International
Class: |
G01N
33/487 (20060101); G01N 33/74 (20060101); G01N
33/78 (20060101); G21h 005/02 () |
Field of
Search: |
;250/83R,83SA,16T
;23/23B |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Lawrence; James W.
Assistant Examiner: Willis; Davis L.
Claims
What is claimed is:
1. A process for the in vitro determination of thyroxine in serum
comprising:
A. adding a predetermined quantity of serum to be tested and a
predeterminate radioactive thyroxine solution to a column packed
with a dextran gel crosslinked with epichlorohydrin and having a
water regain of from 1 to 5 grams per gram of dry gel at a pH of at
least about 11 and allowing the serum and thyroxine to flow into
the column;
B. washing the column with an aqueous alkaline solution;
C. adding a predetermined quantity of an eluting agent containing
thyroxine binding protein to partially remove the thyroxine on the
column;
D. determining the ratio of radioactive thyroxine retained by the
column to that originally added, and
E. calculating the thyroxine content of the serum by comparing the
percent retention to that obtained with known concentrations of
thyroxine in serum.
2. A process as in claim 1 wherein the radioactivity of the gel
column is determined before and after elution to determine the
ratio of radioactive thyroxine retained by the column to that
originally added.
3. A process as in claim 1 wherein the aqueous alkaline solution is
buffered to a pH of about 8 to 10.
4. A process as in claim 3 wherein the aqueous alkaline solution is
a barbital buffer having a pH of 8.6.
5. A process as in claim 1 wherein the pH of the column is
maintained at about 11 to 13.
6. A process as in claim 1 wherein the eluting agent is serum.
7. A process as in claim 1 wherein the eluting agent is human
.alpha.-globulin.
Description
BACKGROUND OF THE INVENTION
The thyroid gland is extremely important in the animal body because
of its effect upon the basal metabolic rate. This effect is
regulated by the thyroxine hormone which is released in response to
nervous or hormonal stimuli. Thyroxine enters the circulatory
system and acts either directly upon the cell or indirectly upon
other hormonal systems. Abnormal activity of the thyroid is a
common malady in humans. In hypothyroidism, the body has decreased
thyroid activity which is manifest by such diseases as cretinism
and myxedema. Hyperthyroidism is a state of excessive thyroid
activity in which one becomes nervous, develops an increased pulse
rate and sometimes goiter.
Thyroid deficiency was associated with a reduced metabolic rate as
early as 1895 and several systems based on basal metabolic rate
were devised for estimating thyroid activity. However, such systems
were not reliable, so more direct and precise methods were sought.
In 1896, iodine was discovered in thyroid extract but the
relationship between blood iodine level and thyroid function was
not firmly established until 1933. This led to the use of
protein-bound iodine as a means of estimating thyroid function, and
by 1955 the PBI test was standard for checking thyroid activity. It
soon became apparent that this test was influenced by the
administration of other iodine containing compounds to the patient.
Thus, the butanol-extractable iodine (BEI) procedure was devised
which gave better correlation between serum iodine levels and the
clinical findings but was likewise non-specific for T-4.
A major advance in T-4 analysis occurred in 1959 when Galton et
al., Biochem. J. 72, 310 (1959) liberated T-4 from serum protein by
hydrolysis and separated it from other iodine compounds by passage
through a resin column. Later, Pileggi et al., J. Clin. Endocr.
Met. 21, 1272 (1961) developed this column chromatographic
procedure into a clinically usable method.
In 1964, Murphy et al., J. Clin. Endocr. Met. 24, 187 (1964)
developed a T-4 assay based upon competitive protein binding and
isotopic dilution which required the initial extraction of T-4 from
the serum with alcohol followed by centrifugation and drying.
Although this test was highly specific, only 80 percent of the T-4
could be recovered from the serum.
More recently, U.S. Pat. No. 3,471,553 set forth still another
column chromatography T-4 assay in which an anion-exchange resin is
adjusted to an alkaline pH of about 12 to dissociate the thyroxine
from its albumin and globulin carriers. The diluted serum solution
is then poured onto the resin wherein proteins, amino acids,
thyroxine, iodotyrosine and inorganic iodine are adsorbed.
Successive washes with acetate buffer isopropyl alcohol and acetic
acid remove serum proteins, iodothyronines and some organic iodine
compounds. Further treatment of the resin with 50 percent acetic
acid at a pH of 2 quantitatively removes T-4. Even though many
modifications have been made in the original T-4 by column assay of
Galton et al., the procedure is substantially the same i.e., a
diluted serum sample is applied to an ion exchange resin. The
unwanted contaminants are then eluted from the column and
discarded. Following this, the hormones to be measured are eluted,
collected and the T-4 determined by iodine analysis.
SUMMARY OF THE INVENTION
The present invention for determining T-4 is based on the
competitive protein binding principle which is a modified form of
saturation analysis. The T-4 to be determined is mixed with a
determinate sample of T-4 labeled with a trace amount of
radioactive isotope. A binding agent is added which will bind a
definite number of molecules. Since the binding agent cannot
distinguish between the labeled and unlabeled molecules, they
compete with each other on an equal basis for the binding sites.
These molecules are uniformly mixed so that the binding agent will
bind them in the same ratio as that existing in the free or unbound
state. As the concentration of the unlabeled molecules increases,
the ratio becomes smaller and fewer labeled molecules are bound by
the binding agent, leaving more of the labeled molecules in the
free state. By calibrating the binding of labeled molecules in the
presence of a known amount of unlabeled molecules, a quantitative
procedure can be established.
In practicing the present invention, the crosslinked dextran gel
column acts as the secondary binding agent for the unbound or free
T-4, whereas the primary binding agent is a thyroxine (T-4) binding
protein. First, a measured amount of serum is mixed with some T-4
labeled with radioactive iodine on top of the column. Both the
column and T-4 mixture are at pH12 to 13. At this pH, the T-4
binding serum proteins such as prealbumin, albumin and thyroxine
binding globulin are completely dissociated from T-4. As the
mixture flows down the column, the T-4 is bound by the dextran gel.
The serum proteins are washed away with a barbital buffer at a pH
of 8.6 which automatically adjusts the pH of the column to that of
the buffer. The pH is such that the transfer of the T-4 from the
column to the primary binding agent is facilitated. The primary
binding agent dissolved in barbital buffer is then added.
Equilibrium is quickly established between the two binding agents
and the primary binder is washed away carrying a portion of the T-4
with it. By measuring the amount of radioactivity on the column
before and after treatment with the primary binding agent and
comparing the percent retained by the column with a standard curve
in which percent retained is plotted against T-4 concentration, the
amount of T-4 in the serum can be determined.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
This invention is predicated upon the discovery that a dextran gel
column retains radioactive T-4 very well up to a pH of 9. Between
pH 9 and 11, radioactive T-4 is rapidly eluted from the column
whereas above pH 11 the retention of T-4 by the column is greatly
enhanced. Thus, dextran gel binds T-4 but not protein, whereas
anion exchange resins bind both T-4 and protein which are removed
nonspecifically and only under acidic conditions.
The dextran gels employed in the column are crosslinked with
various amounts of epichlorohydrin as described in U.S. Pat. No.
3,042,667 and have a water regain of from 1 to 5 grams per gram of
dry gel product. Such gels are produced commercially by Pharmacia
of Uppsala, Sweden and sold under the trade name of Sephadex in
various molecular weight ranges and sieve sizes. Thus, Sephadex
G-10 has a water regain of one gram per gram of dry gel, Sephadex
G-15 has a water regain of 1.5 grams per gram of dry gel, Sephadex
G-25 has a water regain of 2.5 grams per gram of dry gel and
Sephadex G-50 has a water regain of 5 grams per gram of dry gel. Of
these gels, Sephadex G-25 is preferred.
The dextran gel column employed in this invention is prepared by
suspending 500 grams of dry gel in two liters of distilled water
and allowing it to hydrate overnight. Fines are removed by
slurrying the gel in 0.1 N sodium hydroxide for about 5 minutes,
allowing it to settle for 15 minutes and then drawing off the
supernatant by suction. The process is repeated three times and the
gel is suspended in 4,400 milliliters of 0.1 N sodium hydroxide.
Four milliliters of this suspension is placed in a six milliliter
plastic syringe barrel having a diameter of 13 millimeters and a
length of 66 millimeters. Each barrel is prefitted with a bottom
closure means, for example, a removable cap, and a detergent
treated sintered polyethylene retaining disc about 1.5 millimeters
thick and having a diameter of 13 millimeters is pressed coaxially
to the bottom of the plastic barrel. After placement of the
suspension in the syringe barrel, the suspension is stirred and
permitted to settle free of air bubbles after which another
detergent-treated sintered polyethylene disc, like the
first-mentioned disc, is inserted into the syringe barrel and
pushed coaxially into firm contact with the gel. About 1.5
milliliters of sodium hydroxide solution remains above the upper
disc. The upper end of the syringe is closed with a new
polyethylene cap. This procedure provides a column containing about
450 milligrams of gel.
The T-4 test herein contemplated is performed by utilizing the gel
column thus prepared as follows:
1. Remove the top cap from the column, decant the supernatant and
place the column in an upright position.
2. Add 0.45 milliliter of a 0.1 N sodium hydroxide solution
containing about 0.10 microcuries of radioactive T-4 to provide
from 60,000 to 120,000 counts per minute of gamma
radioactivity.
3. Add 0.1 milliliter of serum sample and mix with the radioactive
T-4. If a standard is to be determined, nonradioactive T-4 is
added.
4. Remove the bottom cap and allow the serum mixed with the
radioactive T-4 to flow into the column.
5. Wash the column with 4 milliliters of a 0.075 molar aqueous
barbital solution buffered to a pH of 8.6.
6. Replace the bottom cap and determine the radioactivity of the
column in counts per minute with a gamma counter.
7. Remove the bottom cap and add one milliliter of 0.15% human
.alpha.-globulin dissolved in 0.075 molar barbital buffer.
8. Add 4 milliliters of 0.075 molar barbital buffer at pH 8.6 and
allow to flow through the column.
9. Replace the bottom cap and again determine the radioactivity of
the column.
10. Calculate the percentage of radioactivity retained on the
column and determine the T-4 content of the serum sample by
comparing the percent retention to a standard curve prepared with
T-4 solutions of known concentrations.
Although a specific embodiment of the test method comprising this
invention has been described, it should be understood that several
variations are possible. Thus, the gel column can be made alkaline
by potassium hydroxide or ammonium hydroxide if desired. Human
.alpha.-globulin can be replaced with an equivalent amount of human
serum, bovine serum or bovine gamma globulin as the primary binding
agent. Aqueous alkaline solutions buffered to a pH of from 8 to 10
with sodium phosphate or tris (hydroxymethyl) amino methane can be
used at concentrations from 0.01 to 0.2 molar, but an aqueous
barbital solution is preferred, since it facilitates better
quantitation when used to dissolve the human .alpha.-globulin or
other binding agents.
Another variation of the present invention involves determining the
radioactivity of the solutions before addition to the gel column
and after elution therefrom rather than determining the
radioactivity of the column itself. Percent retention is then
determined by a difference calculation. However, for the sake of
efficiency, it is preferable to determine the radioactivity of the
column before and after elution.
Assays for T-4 by the column methods of the prior art should not be
confused with the present method which involves saturation analysis
using a radioactive tracer. Previously, a column was utilized only
to separate contaminating iodines prior to analysis of iodine. In
certain cases, this was done by using an ion exchange resin column
at an alkaline pH, and iodine analysis was performed on the T-4
recovered from the column by measuring the effect of iodine on the
ceric-arsenious acid reaction. The saturation analysis method
herein disclosed is a direct determination of thyroxine, rather
than the indirect measurement of thyroxine as iodine.
* * * * *