A procedure for the determination of plasma thyroxine in farm animals

ISHII Tadao, TANABE Yuichi, TAMAKI Yoshinori, SHODA Yoichi

doi:10.2508/chikusan.42.216

In the previous report⁵⁾ the availability of the acetalized polyvinyl alcohol (PVF) sponge for the purpose of radiostereo-assay of plasma thyroxine was described. But using PVF sponge for radiostereo-assay of plasma thyroxine instead of Triosorb resin, which was first proposed by NAKAJIMA et al.^{7, 8)}, lowered or sometimes minus value was obtained in animal plasma. Then, we re-examined the extraction procedure of plasma thyroxine (Table 1, 2). It was shown that n-propanol, iso-propanol, ethanol, acetone, dichloroethane and ethyleneglycol-monomethyl-ether were suitable for the extraction of plasma thyroxine (Table 3). Considering the coagulation of protein and volatility, ethanol seemed to be the most suitable solvent to extract the plasma thyroxine. The clearest supernatant was obtained when double or treble volume of ethanol (above 99%) was added to one volume of the plasma. However, 2 ml of ethanol supernatant, which was obtained by adding 2 ml of ethanol to I ml of bovine plasma followed by mixing and centrifuging 3, 000 r.p.m. (1500 x g) for 15 minutes, still contained 1.42 mg of plasma protein maintaining special binding affinity for thyroxine (Fig. 2). After the evaporation, the dried extract was added to ¹³¹I-T₃ sponge uptake test system. Then, the ¹³¹I-T₃ sponge uptake ratio was lowered remarkably (Fig. 1). So the contamination of plasma protein in extracts was the main factor of lowered estimation of plasma thyroxine. Contaminating protein was removed by re-extraction procedure. The ethanol supernatant was evaporated to dryness in a test tube in an electric drying oven at 60°C. To the dried residue 0.1ml of dist. water and 2ml of ethanol were added, and the extraction and drying were repeated. The re-extracted supernatant contained 18.3 pg of protein, which had not the specific binding affinity for thyroxine and did not lower ¹³¹I-T₃ sponge uptake. Thyroxine recovery rate by re-extraction was smaller than that of single-extraction (Fig. 3). The estimated value of plasma thyroxine by re-extraction was higher than that of singleextraction (Fig. 4). It was concluded that ethanol extract, which was obtained by the addition of ethanol twice as much as animal plasma, contained considerable amount of plasma protein. And the contaminating protein maintained specific binding affinity for thyroxine. Then, to remove it for exact estimation of plasma thyroxine in animals, the ethanolreextraction procedure is recomended.

A procedure for the determination of plasma thyroxine in farm animals

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