Test composition and method for the in vitro determination of thyroid function

[57] 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.

Claims, No Drawings

3,716,632

TEST COMPOSITION AND METHOD FOR THE IN VITRO DETERMINATION OF THYROID

FUNCTION

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 crosslinked dextran (Sephadex) is provided to take up any of the added radioactive triiodothyronine or thyroxine (hereinafter referred to as T₃ or T₄, 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 T₃ to the free iodide form, combined with considerable adsorption of the aqueous buffered radioactive T₃ 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 T₃ and (3) decreasing the adsorption of radioactive iodinated T₃ 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 T₃ solution prior to combining with the serum sample or by adding same to the combined T₃ 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 concentra¹⁰ tion of about 5. to 70 micrograms per milliliter of aqueous I^12S-T₃ 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 ¹⁵ 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 ₂₀ changes color from yellow to purple within the pH ⁰ range of 5.2 to 6.8. When mixed with aqueous radioactive T₃ 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. 25 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 30 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 yel³⁵ low 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 T₃ 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 l‘“ 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 ⁵⁵ I^,25-T3 employed in the T3 reagent. This is important, since the gamma counters cannot distinguish between I^,25-T3 and free I¹²⁵, and all of the dose I’²⁵ is counted initially during the test procedure. Washing with water removes any free I¹²⁵ 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 T₃ ratio is subject to a significant error of from 5 percent to 10 percent.

In a series of tests, a buffered aqueous solution of I¹²⁵—T₃ containing 60 micrograms of bromcresol purple per milliliter prepared as shown in the foregoing example was stored under various conditions in a polyethylene

3,716,632 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 T₃ 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° C., the T₃ 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 T₃ test solution is to inhibit the adsorption of radioactive T₃ 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 T₃ 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 -T₃ 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 T₃ employed in the test compositions of this invention can be made radioactive with either I¹²⁵ or I¹³¹, although I¹²⁵ is preferred, since it has a half-life of 56 days compared to I¹³¹ which has a half-life of 8 days.