US3823001A - Determination of thyroid hormone uptake - Google Patents
Determination of thyroid hormone uptake Download PDFInfo
- Publication number
- US3823001A US3823001A US00126316A US12631671A US3823001A US 3823001 A US3823001 A US 3823001A US 00126316 A US00126316 A US 00126316A US 12631671 A US12631671 A US 12631671A US 3823001 A US3823001 A US 3823001A
- Authority
- US
- United States
- Prior art keywords
- absorbent
- column
- thyroid hormone
- serum
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical class IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 title abstract description 49
- 239000005495 thyroid hormone Substances 0.000 title abstract description 28
- 229940036555 thyroid hormone Drugs 0.000 title abstract description 28
- 239000002250 absorbent Substances 0.000 abstract description 38
- 230000002745 absorbent Effects 0.000 abstract description 38
- 210000002966 serum Anatomy 0.000 abstract description 25
- 230000027455 binding Effects 0.000 abstract description 15
- 239000003957 anion exchange resin Substances 0.000 abstract description 11
- 239000000126 substance Substances 0.000 abstract description 3
- 238000000034 method Methods 0.000 description 44
- 239000003456 ion exchange resin Substances 0.000 description 10
- 229920003303 ion-exchange polymer Polymers 0.000 description 10
- 102000004506 Blood Proteins Human genes 0.000 description 9
- 108010017384 Blood Proteins Proteins 0.000 description 9
- 239000012530 fluid Substances 0.000 description 8
- 210000001685 thyroid gland Anatomy 0.000 description 8
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical group C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 7
- 239000011347 resin Substances 0.000 description 7
- 229920005989 resin Polymers 0.000 description 7
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 229940088597 hormone Drugs 0.000 description 5
- 239000005556 hormone Substances 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 229940034208 thyroxine Drugs 0.000 description 5
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 description 5
- 229940035722 triiodothyronine Drugs 0.000 description 5
- 230000014759 maintenance of location Effects 0.000 description 4
- 230000002285 radioactive effect Effects 0.000 description 4
- 108091023025 thyroid hormone binding Proteins 0.000 description 4
- 102000028501 thyroid hormone-binding Human genes 0.000 description 4
- 241000736355 Euthyroides Species 0.000 description 3
- 230000009137 competitive binding Effects 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 206010020850 Hyperthyroidism Diseases 0.000 description 2
- 102000023732 binding proteins Human genes 0.000 description 2
- 108091008324 binding proteins Proteins 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical group Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 235000006679 Mentha X verticillata Nutrition 0.000 description 1
- 240000003321 Mentha requienii Species 0.000 description 1
- 235000002899 Mentha suaveolens Nutrition 0.000 description 1
- 235000001636 Mentha x rotundifolia Nutrition 0.000 description 1
- 208000024799 Thyroid disease Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- -1 anion ion Chemical class 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 229940023913 cation exchange resins Drugs 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000003610 charcoal Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000009790 rate-determining step (RDS) Methods 0.000 description 1
- 239000012508 resin bead Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 229940032953 thyroid therapy drug Drugs 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/804—Radioisotope, e.g. radioimmunoassay
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/825—Pretreatment for removal of interfering factors from sample
Definitions
- This invention relates to the clinical determination of thyroid function. More particularly, it relates to the determination of thyroid hormone uptake by a secondary binding substance or absorbent from which an estimation of the concentration of available thyroid protein hormone binding sites in blood can be made.
- the invention involves disposing a competitive binding absorbent for thyroid hormone in a column.
- a fluid sample containing thyroid hormone bound by thyroid hormone binding proteins is flowed through the column.
- the column dimensions and absorbent are selected to competitively bind and retain about -90 weight percent of the bound thyroid hormone in the fluid sample during a single passage of the sample through the column.
- The. amount of thyroid hormone retained in the column relative to the thyroid hormone present in the column efiluent is then measured.
- T-4 thyroxine
- T-3 tri-iodothyronine
- the degree of saturation of the serum proteins by thyroid hormone varies in different states of thyroid function.
- the serum proteins are saturated with thyroid hormones to greater extent and therefore the number of available binding sites on serum proteins (unsaturated thyroid-hormone-binding capacity) is decreased.
- hypothyrodism Since thyroxine is bound to serum proteins more firmly than tri-iodothyronine, the number of available binding sites on serum proteins depends mainly on the concentration of thyroxine in plasma.
- Hamolski et al., J. Clin. Endocr. 17, 33 (1957) described the uptake of I-131 labeled tri-iodothyronine by red cells in whole blood as a method for estimating the saturation of thyroid binding proteins. They discovered that I-l3l labeled tri-iodothyronine incubated with plasma from hyperthyroid patients demonstrated a lower rate of absorbence tf radioactivity onto the erythrocytes as compared with euthyroid patients. This method has proved valuable in the assessment of thyroid diseases, abnormalities of thyroxine transporting protein, and in the evaluation of thyroid therapy. Since the measurement is performed using a radioactive labeled hormone, interferences from serum organic iodine contaminants is minimized.
- these T-3 uptake tests are performed by equilibrating trace amounts of radioactively labeled T-3 with a sample of patients serum. This combination is then equilibrated with a secondary binding material such as red blood cells.
- the secondary absorbent has a certain aifinity for the T-3 so that some of the T 3 will be-ab 3,823,001 Patented July 9, 1974 sorbed by this secondary binding material. If the absorbent has a fixed aflinity for T-3, the distribution of the radioactive T-3 between the absorbent and the serum proteins will depend upon the number of available binding sites on the serum proteins.
- absorbents Due to various inherent difficulties in using red blood cells, a variety of other absorbents have been utilized.
- One such absorbent is a strong base anion ion exchanger which has been utilized in the form of free resin beads, resin imbedded in a sponge, and as a membrane.
- Another material that has been used is hemoglobin absorbed on charcoal. Such materials have the ability to remove T-3 from the binding proteins.
- the new column procedure provides for a residence time of contact between the sample and the absorbent of not more than about 10 minutes and most commonly not more than about 5 minutes. In the preferred embodiment to be described hereinafter the residence time is less than about three minutes. At the conclusion of the residence period the liquid phase is directly obtained as column effluent separated from the solid phase absorbent so that there is no need for a separation step as in the previously used batch technique.
- the residence time is relatively very brief, comparable or better results than that obtained with prior techniques are achieved. This is most surprising when considering the efforts previously put forth in terms of continuous mixing for extended periods of time in order to obtain or approach a batch'equilibrium condition.
- the present method has been compared with the prior art method using a batch equilibrium procedure and a straight line correlation between the two methods was obtained. In only 2% of the cases tested was a result obtained by the present method which diflered from that of the prior batch technique.
- the present method provides a measurement related to the rate of dissociation of T-3 from its protein binding sites.
- conditions have been adjusted so that an equilibrium condition or one approaching equilibrium was obtained.
- Such test results provide only a measurement of the equilibrium binding capacity of the binding sites.
- the results are related to the rate of T-3 uptake by the absorbent, the T-3 binding capacity of the proteins, and the rate at which these proteins dissociate with T-3. Since the rate of dissociation of T-3 from the protein is the rate determining step, such rate is the one primarily measured by the present method.
- any suitable absorbent may be used which under the conditions of the test absorb about -90 weight percent of the thyroid hormone in the test sample.
- the absorbent and the other test conditions are selected so that about 40-60 weight percent of the thyroid hormone from a serum sample of a euthyroid patient are absorbed.
- a preferred class of absorbents for use in the present invention are ion exchange resins.
- Anion exchange resins are particularly desirable where a radioactively labeled thyroid hormone is utilized for determining the percentage of uptake by the absorbent.
- the anion exchange resin serves to remove any radioactive breakdown products which might otherwise interfere with the test results.
- a strong base anion exchange resin provides particularly desirable results.
- a preferred procedure for accomplishing this is to include a preselected amount of radioactively labeled T-3 4 taining 8%. by weight of cross-linkage, having a 100- 200 mesh size, and in a column diameter of about 0.75 centimeters. Using such a column the residence time of the sample in the column is not more than about three mintes.
- the fluid sample being tested will contain up to about 1 ml. of blood serum and preferably about .5 ml. of blood serum. Consistent with the prior art, the sample is conveniently diluted with a buffer solution preferably having a pH of about 5.
- EXAMPLE 0.5 ml. of patients serum is mixed with 2.5 mls. of 0.4 N sodium acetate buffer at pH 5 containing trace amount of radioactively labeled tri-iodothyronine. This mixture is allowed to sit for 20 minutes to insure random distribution of the radioactive T-3 between the serum and solution. This mixture is then passed over a strong base anion exchange resin in a column.
- the column contains 1.2 ml. of a strong base anion exchange resin in the chloride form of 8% cross-linkage and 100-200 mesh.
- the internal diameter of the column is about 0.75 cm. diameter.
- the efliuent from the column is collected in a counting tube. The contents of the tube are then measured for the quantity of radioactivity. By comparison with a control such as normal pooled serum or commercial control serum of known value, an evaluation of the degree of unsaturation of the serum protein binding sites can be made.
- No'rE.TBI value for abnormal serum was calculated to be .68 from charted results using the prior art batch method.
- AG Resins are ion exchange resins commercially available from Bio-Rad Laboratories, Richmond, California.
- AGl Resins are strong base anion exchang resins.
- AGSOW Resins are strongly acidic cation exchange resins. The number following the X" indicates the weight percent of cross-linkage in the resin.
- the sample is passed through the column and the amount of radioactively labeled T-3 or T-4 in the efiiuent is measured by conventional techniques.
- the values obtained for uptake of the radioactively labeled hormone enable thyroid function to be evaluated by reference to values from known serum processed under comparable conditions.
- the desired information can be obtained by measuring the amount of radioactivity in the absorbent or in the column eflluent. Preferably, the measurements are made on the efiluent.
- a method for determining the uptake of thyroid hormone by a competitive binding absorbent therefor from a fluid sample containing thyroid hormone bound by thyroid hormone binding proteins comprising: disposing said absorbent in a column, flowing said fluid sample through and out of said column at a constant rate, said column dimensions and absorbent being selected to competitively bind and retain about 10-90 weight percent of the bound thyroid hormone in said fluid sample during a single passage therethrough, measuring the amount of thyroid hormone retained in said column relative to the amount present in the eflluent therefrom, and evaluating the uptake of thyroid hormone from said sample by comparison with a control.
- An improved clinical procedure for evaluating thyroid status comprising: equilibrating a sample containing blood serum with a preselected amount of radioactively labeled thyroid hormone in a buffer diluent, providing a column of ion exchange resin adapted to absorb about 10-90 weight percent of the serum bound thyroid hormone in said sample, passing said equilibrated sample and diluent through said column at a constant rate, passage through the column being completed in not more than about five minutes, measuring the radioactivity of said column and/or the efiluent therefrom, determining the percentage of thyroid hormone absorbed by said column and/or remaining in said elfiuent from said radioactivity measurement, and evaluating thyroid status of said sample by comparison with a control.
- radioactively labeled thyroid hormone is radioactively labeled tri-iodothyronine.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Endocrinology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
DETERMINATION OF THYROID HORMONE UPTAKE FROM BLOOD SERUM BY A SECONDARY BINDING SUBSTANCE OR ABSORBENT IS EXECUTED BY FLOWING A SERUM SAMPLE THROUGH A COLUMN CONTAINING THE ABSORBENT IN A VERY BRIEF PERIOD. A RESIDENCE TIME OF THREE MINUTES OR LESS IS TYPICAL WHEN USING A CROSS-LINKED STRONG BASE ANION EXCHANGE RESIN AT THE ABSORBENT.
Description
United States Patent 3,823,001 DETERMINATION OF THYROID HORMONE UPTAKE Burton A. Zabin, Berkeley, Calif., assignor to Bio-Rad Laboratories, Richmond, Calif. No Drawing. Filed May 6, 1971, Ser. No. 126,316 Int. Cl. G01n 33/ 16' US. Cl. 23230 B 14 Claims ABSTRACT OF THE DISCLOSURE Determination of thyroid hormone uptake from blood serum by a secondary binding substance or absorbent is executed by flowing a serum sample through a column containing the absorbent in a very brief period. A residence time of three minutes or less is typical when using a cross-linked strong base anion exchange resin as the absorbent.
This invention relates to the clinical determination of thyroid function. More particularly, it relates to the determination of thyroid hormone uptake by a secondary binding substance or absorbent from which an estimation of the concentration of available thyroid protein hormone binding sites in blood can be made.
Briefly, the invention involves disposing a competitive binding absorbent for thyroid hormone in a column. A fluid sample containing thyroid hormone bound by thyroid hormone binding proteins is flowed through the column. The column dimensions and absorbent are selected to competitively bind and retain about -90 weight percent of the bound thyroid hormone in the fluid sample during a single passage of the sample through the column. The. amount of thyroid hormone retained in the column relative to the thyroid hormone present in the column efiluent is then measured.
By way of background, it is known that thyroxine (T-4) and tri-iodothyronine (T-3) in plasma are bound by dissociable linkages to specific serum proteins collectively referred to as thyroid hormone binding proteins.
The degree of saturation of the serum proteins by thyroid hormone varies in different states of thyroid function. In hyperthyroidism, forexample, the serum proteins are saturated with thyroid hormones to greater extent and therefore the number of available binding sites on serum proteins (unsaturated thyroid-hormone-binding capacity) is decreased. The converse is true in hypothyrodism. Since thyroxine is bound to serum proteins more firmly than tri-iodothyronine, the number of available binding sites on serum proteins depends mainly on the concentration of thyroxine in plasma.
Hamolski et al., J. Clin. Endocr. 17, 33 (1957) described the uptake of I-131 labeled tri-iodothyronine by red cells in whole blood as a method for estimating the saturation of thyroid binding proteins. They discovered that I-l3l labeled tri-iodothyronine incubated with plasma from hyperthyroid patients demonstrated a lower rate of absorbence tf radioactivity onto the erythrocytes as compared with euthyroid patients. This method has proved valuable in the assessment of thyroid diseases, abnormalities of thyroxine transporting protein, and in the evaluation of thyroid therapy. Since the measurement is performed using a radioactive labeled hormone, interferences from serum organic iodine contaminants is minimized.
In general, these T-3 uptake tests are performed by equilibrating trace amounts of radioactively labeled T-3 with a sample of patients serum. This combination is then equilibrated with a secondary binding material such as red blood cells. The secondary absorbent has a certain aifinity for the T-3 so that some of the T 3 will be-ab 3,823,001 Patented July 9, 1974 sorbed by this secondary binding material. If the absorbent has a fixed aflinity for T-3, the distribution of the radioactive T-3 between the absorbent and the serum proteins will depend upon the number of available binding sites on the serum proteins.
Due to various inherent difficulties in using red blood cells, a variety of other absorbents have been utilized. One such absorbent is a strong base anion ion exchanger which has been utilized in the form of free resin beads, resin imbedded in a sponge, and as a membrane. Another material that has been used is hemoglobin absorbed on charcoal. Such materials have the ability to remove T-3 from the binding proteins.
In all of the prior competitive binding procedures the absorbent and the serum sample have been contacted on a batch basis as distinguished from the column technique of this invention. In these prior procedures an equilibrium condition or a condition approaching equilibrium has been sought because it has been felt that clinically reproducible results could only be achieved in that way. To this end the serum sample and the absorbent were constantly mixed and moved as by shaking or turning for a relatively extended period of time varying from about 30 minutes to about an hour and a half. At the end of the mixing period a separation of the absorbent and the liquid phase was necessary in order to determine the percentage of uptake by the absorbent. Care was needed to obtain uniformity in the mixing time with the samples being tested and the controls used to obtain useful known values for the test procedure.
In accordance with the present procedure, these disadvantageous steps and long time periods are eliminated. In general, the new column procedure provides for a residence time of contact between the sample and the absorbent of not more than about 10 minutes and most commonly not more than about 5 minutes. In the preferred embodiment to be described hereinafter the residence time is less than about three minutes. At the conclusion of the residence period the liquid phase is directly obtained as column effluent separated from the solid phase absorbent so that there is no need for a separation step as in the previously used batch technique. Despite the fact that the residence time is relatively very brief, comparable or better results than that obtained with prior techniques are achieved. This is most surprising when considering the efforts previously put forth in terms of continuous mixing for extended periods of time in order to obtain or approach a batch'equilibrium condition.
In the present column technique no equilibrium condi tion between absorbent and solution exists. Instead a constantly changing relationship between absorbent and solution is presented as the solution moves through the column absorbent. In a sense the results from the present technique are generated by a series of initial uptakes of thyroid hormone by successive portions of absorbent as the solution moves through the column. At the point of each initial uptake the slope of an uptake curve which can be drawn is so rapidly changing that an acceptable degree of reproducibility of results would not be expected. Nevertheless, it has been found that the present column technique provides results comparable or better than the batch technique. For example, 17 portions of the same sample of serum have been processed in a column in accordance with the present method and the percent error was 2.5%. This percent error is better than most prior art methods and quite acceptable for clinical diagnostic purposes. 'In addition, the present method has been compared with the prior art method using a batch equilibrium procedure and a straight line correlation between the two methods was obtained. In only 2% of the cases tested was a result obtained by the present method which diflered from that of the prior batch technique.
Apart from its simplicity and time savings, another feature believed to be oflered by the present method is that it provides a measurement related to the rate of dissociation of T-3 from its protein binding sites. In the prior batch procedures conditions have been adjusted so that an equilibrium condition or one approaching equilibrium was obtained. Such test results provide only a measurement of the equilibrium binding capacity of the binding sites. In the present method when applied to T-3 uptake, it is believed that the results are related to the rate of T-3 uptake by the absorbent, the T-3 binding capacity of the proteins, and the rate at which these proteins dissociate with T-3. Since the rate of dissociation of T-3 from the protein is the rate determining step, such rate is the one primarily measured by the present method.
In practicing the present method any suitable absorbent may be used which under the conditions of the test absorb about -90 weight percent of the thyroid hormone in the test sample. Preferably the absorbent and the other test conditions are selected so that about 40-60 weight percent of the thyroid hormone from a serum sample of a euthyroid patient are absorbed. A preferred class of absorbents for use in the present invention are ion exchange resins. Anion exchange resins are particularly desirable where a radioactively labeled thyroid hormone is utilized for determining the percentage of uptake by the absorbent. The anion exchange resin serves to remove any radioactive breakdown products which might otherwise interfere with the test results. As will be seen in the examples hereinafter, a strong base anion exchange resin provides particularly desirable results.
Any technique for analytically measuring the percentage uptake of hormone by the absorbent is contemplated. A preferred procedure for accomplishing this is to include a preselected amount of radioactively labeled T-3 4 taining 8%. by weight of cross-linkage, having a 100- 200 mesh size, and in a column diameter of about 0.75 centimeters. Using such a column the residence time of the sample in the column is not more than about three mintes.
Preferably the fluid sample being tested will contain up to about 1 ml. of blood serum and preferably about .5 ml. of blood serum. Consistent with the prior art, the sample is conveniently diluted with a buffer solution preferably having a pH of about 5.
To illustrate the invention, the following procedure has been shown to give excellent results.
EXAMPLE 0.5 ml. of patients serum is mixed with 2.5 mls. of 0.4 N sodium acetate buffer at pH 5 containing trace amount of radioactively labeled tri-iodothyronine. This mixture is allowed to sit for 20 minutes to insure random distribution of the radioactive T-3 between the serum and solution. This mixture is then passed over a strong base anion exchange resin in a column. The column contains 1.2 ml. of a strong base anion exchange resin in the chloride form of 8% cross-linkage and 100-200 mesh. The internal diameter of the column is about 0.75 cm. diameter. The efliuent from the column is collected in a counting tube. The contents of the tube are then measured for the quantity of radioactivity. By comparison with a control such as normal pooled serum or commercial control serum of known value, an evaluation of the degree of unsaturation of the serum protein binding sites can be made.
The first line in Table I below indicates the results obtained with the materials described in the preceding paragraph. The data shown in lines 2-5 of the table were developed using the same procedure but different ion exchange resins and volumes as indicated.
TABLE I.-EFFECT OF RESIN TYPE ON PERFORMANCE Control serum, percent eluted l TBI=Thyroid Binding Index.
No'rE.TBI value for abnormal serum was calculated to be .68 from charted results using the prior art batch method. AG Resins are ion exchange resins commercially available from Bio-Rad Laboratories, Richmond, California. AGl Resins are strong base anion exchang resins. AGSOW Resins are strongly acidic cation exchange resins. The number following the X" indicates the weight percent of cross-linkage in the resin.
or T-4 in the serum sample. After equilibrium has been attained the sample is passed through the column and the amount of radioactively labeled T-3 or T-4 in the efiiuent is measured by conventional techniques. As understood in the art, the values obtained for uptake of the radioactively labeled hormone enable thyroid function to be evaluated by reference to values from known serum processed under comparable conditions. The desired information can be obtained by measuring the amount of radioactivity in the absorbent or in the column eflluent. Preferably, the measurements are made on the efiluent.
In achieving a retention by the absorbent of 10-90 weight percent of the hormone in the sample, a number of variables are involved. The absorbent, its type (i.e. whether or not it is an ion exchange resin including degree of cross-linkage if a resin), volume employed, and mesh size will alfect the percentage retention. In addition, the column dimensions particularly its diameter will bear upon this result. In general, conditions are selected so that with the volume of sample fluid employed the desired extent of retention is achieved within the brief period of time noted. As will be seen in the example to follow, the desired thyroid hormone retention within a brief residence period can be attained using 1.2 ml. (particle volume) of a strong base anion exchange resin con- What is claimed is:
1. A method for determining the uptake of thyroid hormone by a competitive binding absorbent therefor from a fluid sample containing thyroid hormone bound by thyroid hormone binding proteins comprising: disposing said absorbent in a column, flowing said fluid sample through and out of said column at a constant rate, said column dimensions and absorbent being selected to competitively bind and retain about 10-90 weight percent of the bound thyroid hormone in said fluid sample during a single passage therethrough, measuring the amount of thyroid hormone retained in said column relative to the amount present in the eflluent therefrom, and evaluating the uptake of thyroid hormone from said sample by comparison with a control.
2. A method in accordance with claim 1 wherein said column dimensions, absorbent type, volume and mesh size, and the volume of said sample fluid are selected to provide a sample residence time of not more than about ten minutes.
3. A method in accordance with claim 1 wherein said absorbent is an ion exchange resin.
4. A method in accordance with claim 3 wherein said ion exchange resin is a cross-linked anion exchange resin.
5. An improved clinical procedure for evaluating thyroid status comprising: equilibrating a sample containing blood serum with a preselected amount of radioactively labeled thyroid hormone in a buffer diluent, providing a column of ion exchange resin adapted to absorb about 10-90 weight percent of the serum bound thyroid hormone in said sample, passing said equilibrated sample and diluent through said column at a constant rate, passage through the column being completed in not more than about five minutes, measuring the radioactivity of said column and/or the efiluent therefrom, determining the percentage of thyroid hormone absorbed by said column and/or remaining in said elfiuent from said radioactivity measurement, and evaluating thyroid status of said sample by comparison with a control.
6. An improved method in accordance with claim wherein said radioactively labeled thyroid hormone is radioactively labeled tri-iodothyronine.
7. An improved method in accordance with claim 5 wherein said radioactively labeled thyroid hormone is radioactively labeled thyroxine.
8. An improved method in accordance with claim 5 wherein said sample contains a buffer diluent.
9. An improved method in accordance with claim 5 wherein said ion exchange resin is a strong base anion exchange resin.
10. An improved method in accordance with claim 9 wherein said anion exchange resin contains about 8% cross-linkage.
11. An improved method in accordance with claim 5 wherein said column of ion exchange resin is adapted to absorb about -60 weight percent of thyroid hormone from a serum sample of a euthyroid patient.
12. An improved method in accordance with claim 10 wherein said ion exchange resin has a -200 mesh size, is present to provide a bed volume of about 1.2 ml. and a column diameter of about .75 centimeters and the residence time of the sample in the column is not more than about three minutes.
13-. An improved method in accordance with claim 12 wherein said sample contains up to about 1 m1. of blood serum.
14. An improvement in accordance with claim 13 wherein said sample contains about .5 ml. of blood serum.
References Cited UNITED STATES PATENTS 3,206,602 9/1965 Eberle 23-230 B 3,451,777 6/1969 Di Giulio 23230 B 3,659,104 4/1972 Gross et al. 23230 B 3,494,744 5/1972 Zbrowski 23230 B OTHER REFERENCES Hamolsky, et al., J. Clinical Endocrinology & Metabolism, Vol. 17, pp. 33-44 (January 1957).
R. E. SERWIN, Primary Examiner
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US00126316A US3823001A (en) | 1971-05-06 | 1971-05-06 | Determination of thyroid hormone uptake |
CA139,625A CA975269A (en) | 1971-05-06 | 1972-04-13 | Determination of thyroid hormone uptake |
ES402130A ES402130A1 (en) | 1971-05-06 | 1972-04-26 | Determination of thyroid hormone uptake |
DE19722221924 DE2221924C3 (en) | 1971-05-06 | 1972-05-04 | Method of determining the absorption of thyroid hormone |
IT50066/72A IT957786B (en) | 1971-05-06 | 1972-05-05 | APPARATUS FOR DETERMINING THE ABSORPTION OF THYROID CHROMONE |
FR727216152A FR2135658B1 (en) | 1971-05-06 | 1972-05-05 | |
GB2117372A GB1380778A (en) | 1971-05-06 | 1972-05-05 | Determination of thyroid hormone uptake |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US00126316A US3823001A (en) | 1971-05-06 | 1971-05-06 | Determination of thyroid hormone uptake |
Publications (1)
Publication Number | Publication Date |
---|---|
US3823001A true US3823001A (en) | 1974-07-09 |
Family
ID=22424156
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US00126316A Expired - Lifetime US3823001A (en) | 1971-05-06 | 1971-05-06 | Determination of thyroid hormone uptake |
Country Status (6)
Country | Link |
---|---|
US (1) | US3823001A (en) |
CA (1) | CA975269A (en) |
ES (1) | ES402130A1 (en) |
FR (1) | FR2135658B1 (en) |
GB (1) | GB1380778A (en) |
IT (1) | IT957786B (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4015939A (en) * | 1976-05-12 | 1977-04-05 | Bio-Rad Laboratories, Inc. | Competitive binding thyroid assay with improved bound-free separation step |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4301139A (en) * | 1979-06-21 | 1981-11-17 | Ames-Yissum Ltd. | Multilayer column chromatography specific binding assay method, test device and test kit |
-
1971
- 1971-05-06 US US00126316A patent/US3823001A/en not_active Expired - Lifetime
-
1972
- 1972-04-13 CA CA139,625A patent/CA975269A/en not_active Expired
- 1972-04-26 ES ES402130A patent/ES402130A1/en not_active Expired
- 1972-05-05 IT IT50066/72A patent/IT957786B/en active
- 1972-05-05 GB GB2117372A patent/GB1380778A/en not_active Expired
- 1972-05-05 FR FR727216152A patent/FR2135658B1/fr not_active Expired
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4015939A (en) * | 1976-05-12 | 1977-04-05 | Bio-Rad Laboratories, Inc. | Competitive binding thyroid assay with improved bound-free separation step |
Also Published As
Publication number | Publication date |
---|---|
IT957786B (en) | 1973-10-20 |
ES402130A1 (en) | 1975-03-16 |
DE2221924A1 (en) | 1972-11-23 |
DE2221924B2 (en) | 1976-01-22 |
CA975269A (en) | 1975-09-30 |
FR2135658A1 (en) | 1972-12-22 |
FR2135658B1 (en) | 1973-07-13 |
GB1380778A (en) | 1975-01-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
LEVY et al. | Radioimmunoassay of human thyroxine-binding globulin (TBG) | |
Peters et al. | A new method for the determination of serum iron-binding capacity. I | |
US3442819A (en) | Molecular sieve coated particulate adsorbent and processes using same | |
Miller et al. | Anion-exchange chromatographic determination of bromide in serum. | |
US3451777A (en) | Method and apparatus for determining the thyroid hormone content of blood | |
Refetoff et al. | Estimation of the T4 binding capacity of serum TBG and TBPA by a single T4 load ion exchange resin method | |
US3659104A (en) | Method of measuring serum thyroxine | |
JPS6236181B2 (en) | ||
US4225574A (en) | Method for determination of free hormones in biologic fluids | |
US3799740A (en) | Clinical competitive-binding test methods | |
Nolan et al. | Determination of metallothionein in tissues by radioimmunoassay and by cadmium saturation method | |
US3823001A (en) | Determination of thyroid hormone uptake | |
Gallagher et al. | Molecular weight of a purified chicken antibody | |
IL34387A (en) | Blood diagnostic methods employing radioactive tracer compounds and ion-exchange resin membrane | |
US3941564A (en) | Method for assessing thyroid function | |
US3960492A (en) | Method for determining an index of binding protein content of blood | |
US3773467A (en) | Microassay diagnostic test method | |
Schroeder et al. | Microchromatography of hemoglobins. IV. An improved procedure for the detection of hemoglobins S and C at birth | |
US3929981A (en) | Method for determining thyroid function | |
US4478945A (en) | Method for evaluating thyroid function | |
Anthony et al. | The variable influence of an α component in pregnancy plasma on four different assay systems for the measurement of pregnancy-specific β1 glycoprotein | |
Abraham et al. | Microchromatography of hemoglobins: VII. Detection of some uncommon hemoglobin variants and two rapid methods for the quantitation of Hb-A2 in the presence of Hb-C | |
Chaplin et al. | Quantification of antibodies to the C3d subcomponent of human C3. | |
Seligson et al. | Measurement of serum thyroxine-binding capacity | |
Cowley et al. | A radioimmunoassay of human prealbumin in body fluids |