家畜の血漿中サイロキシンの定量法

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タイトル別名
  • A procedure for the determination of plasma thyroxine in farm animals
  • カチク ノ ケッショウ チュウ サイロキシン ノ テイリョウホウ 3 ソクテイ セイド ニ エイキョウ スル ヨウイン ノ カイメイ
  • III. Factors affecting the precision of the determination
  • III. 測定精度に影響する要因の解明

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Using a PVF sponge (acetalized poly-vinyl-alcohol sponge) which has a specific affinity to thyroxine (T4) and tri-iodo-thyronine (T3) in radiostereo-assay, we divised a procedure for measuring plasma-thyroxine in farm animals. The principle of this procedure is that thyroxine-binding protein (TBP) in the plasma binds specifically T4 and T3, and the binding affinity of T4 is greater than that of T3.<br>An unknown amount of T4, extracted from the plasma sample, and a known amount of 131I-labeled T3 were added to the standard plasma solution and the mixture was incubated. After the incubation, the sponge was put into the mixture to adsorb T3 which remained in the solution without binding TBP. When the more T4 was extracted in the plasma sample, the more labeled T3 was adsorbed by the sponge.<br>The amount of extracted T4 was calibrated from the standard curve, which was obtained by the same procedure adding the known amount of T4.<br>After the correction for recovery, the T4 value of the plasma sample was obtained.<br>Practical procedure: One ml of plasma sample and 2ml of ethanol (concentration above 99%) are mixed thoroughly. After allowing the mixture to stand for 5min. at room temperature, the mixture is centrifuged at 3, 000rpm(1, 500×g) for 15min. Two ml of the supernatant is evaporated to dryness in a test tube in an electric drying oven at 60°C. The dried residue is dissoleved in the 0.1ml of distilled water. To the residue, 2ml of ethanol is added, and the extraction and evaporation are repeated again by the same method as the first extraction.<br>The preparation is redissolved in the standard plasma solution which is a mixture of 0.25 ml of bovine plasma, 0.75ml of phosphate buffer (pH8.0, μ=0.1) and 0.1μCi of 131I-T3 (100μCi/ml, 0.5μg/ml).<br>After the incubation of 15min. at 1°C, PVF sponge is added and the mixture is reincubated for 60min. at 1°C.<br>By using a well-type scintillation counter a total count (a cpm) is measured during the incubation period. The radioactivity remaining in the sponge (b cpm) is counted after the sponge is washed with water.<br>131I-T3 sponge uptake value (U%) will be (b/a)×100.<br>Method of drawing standard curve: Ten point three mg of sodium-L-thyroxine is dissolved in 1ml of 0.1N-NaOH and 1ml of propylenglycol, and a further dilution is made with 50v/v% propylenglycol aqueous solution to obtain 10μg/ml T4 solution.<br>Various known volumes of T4 solution (0-50μg) are added to each tube which contains 1ml of the standard plasma solution. According to the above doscribed method, the 131I-T3 sponge uptake value is measured.<br>All the values are plotted on the Y axis against the T4 content on the X axis.<br>Determinatlon of the recovery ratio of T4 in the extract: One tenth μCi of 131I-T4 (100μCi/ml, 5μg/ml) is added to 1ml of T4 in the plasma sample. After measuring of total count (C1 cpm), the mixture is extracted twice by the same procedure which was discribed in the determination of plasma T4.<br>After re-extraction, the radioactivity of the extract is measured (C2 cpm).<br>Recovery ratio of T4 (R%) will be (C2/C1)×100. <br>It is necessary to correct the back ground counts and the physical decay which is going on during the procedure.

収録刊行物

  • 日本畜産学会報

    日本畜産学会報 42 (12), 634-640, 1971

    公益社団法人 日本畜産学会

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