Cellulose Acetate Membrane によるThyroxine-binding Protein の測定法

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A simple method for measurement of thyroxine-binding proteins (TBP) was studied, using cellulose acetate membrane as supporting medium for zone electrophoresis. Electrophoresis was performed at room temperature in Oxoid cellulose acetate membrane. Nine strips, each 2 by 7 cm. in size, were suspended horizontally in a closed system between plastic plates. The ends were connected by filter papers with two vessels, each containing 240 ml. of buffer. Buffers employed were barbital and tris-maleate, both at pH 8.6. In conventional method, thyroxine (T4) -serum mixture was applied in a band, 2 cm. from the cathodalend of the strip and in a reverse-flow method, 1.5cm. from the anodal end. Electric current was kept constant, i.e. 0.4 mA/width of lcm of the strip. All other conditions were the same as the ordinary electrophoresis in the both methods. Time for electrophoresis was 40-50 min. Low background beta counter with anticoincidence circuit was used in the measurement of trace amount of radiothyroxine. The proteins on the strips were stained with Ponceau 3R. A satisfactory separation could be achieved in radioautograph of TBP without distortion of bands. In barbital, when this method is used to measure the affinity of TBP, both conventional and reverse-flow methods can be used. To measure the maximum capacity of thyroxine-binding globulin (TBG), reverse-flow method was necessary. With the conventional method employing tris-maleate buffer, the capacity of TBG and T4-binding prealbumin (TBPA) was not obtained due to trailing of protein-bound T4. Radioactivity measurement of TBP was done before and after staining of proteins in the same normal serum to which was added T4 1.0 and 3.2 μg/ml. T4 distribution of TBP was compared and found not to be altered between nonstained and stained rogups. The result suggested a safe staining procedure not affecting T4 distribution of TBP. T4 distribution among proteins at several levels to which T4 was added was also studied in barbital and tris-maleate buffers, and the mean values for the affinity and capacity of TBP in 18 normal adults by the present method were as follows : The affinity in barbital buffer at added T4 0.002 μg/ml. Conventional : TBG 80%, Albumin 16%. Reverse-flow : TBG 79%, Albumin 18%. In tris-maleate buffer, TBG 67%, Albumin 14%, TBPA 20%. The capacity of TBG in barbital buffer was 0.22±0.03 (Mean±S.D.) μg/ml, with a range of 0.18-0.26 μglml. The capacity of TBG in 10 cases with untreated hyperthyroidism was nearly within normal limits, while in untreated hypothyroidism the capacity was high in 4 out of 6 cases. The above results proved the present method is a simple, rapid and reliable technique for clinical use in place of commonly used paper electrophoresis.
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Cellulose Acetate Membrane によるThyroxine-binding Protein の測定法

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