Abstract
The deiodination of thyroxine has been studied with radioactive iodinated thyroxine. The labeling of 3, 5 positions of the molecule is so difficult that only commercially available radiothyroxine is labeled with 131I at its 3', 5' positions. Therefore, the metabolism of iodines of 3, 5 positions remains to be clarified.
The present study was designed to prepare the doubly labeled thyroxine by mixing individually prepared 3', 5'-125I-thyroxine and 3, 5-131I-thyroxine and to investigate its metabolism.
3, '5'-125I-thyroxine was prepared by a new exchange labeling method. In this procedure, Na125I was oxidized to 125I2 by HCl and H2O2 in a small test tube. The resultant 125I2 was extracted with ethyl ether followed by adding thyroxine in 50% ethanol buffered solution (pH 5.0), in which the exchange reaction took place. The yield was approximately 75%. The specific activity of the labeled thyroxine was 1 mCi/mg.
The same method of labeling was also applied successfully to the preparation of 3, 5-131I-diiodotyrosine which was a starting material of 3, 5-131I-thyroxine.
3, 5-131I-thyroxine was chemically synthesized employing the micro scale coupling reaction of Shiba & Cahnmann with DIHPPA and 3, 5-131I-diiodotyrosine. The product was purified by paperchromatography. The specific activity of the labeled product was 600-800μCi/mg.
Both labeled thyroxines were identified by paperchromatography in three different solvents, high voltage paperelectrophoresis and multiple recrystalizations.
The radioactivity of 125I and 131I was separately measured by a well-type scintillation counter with two γ-ray spectrometers which were set for γ-ray photopeaks of 125I and 131I, respectively.
The deiodination of the doubly labeled radio-thyroxine in homogenates and slices of rat tissues (liver, kindey, muscle, hypophysis and brain) was examined. In all cases the deiodination of 3', 5' positions were more rapid than those of 3, 5 positions.
Significant deiodinations of 3, 5 positions, as well as 3', 5' positions, occurred by exposing to ultra violet of a sterilamp but both deiodinations were inhibited when 5×10-5M thio-uracil, 50% propylene glycohol or 50% human serum were added.
After intravenous administration of the doubly labeled radiothyroxine to rats, urinary excretion of 125I was almost parallel with that of 131I. Paperelectrophoresis proved that 90-95% of the radioactivity was inorganic iodine. Bile was cellected continuously by a biliary fistula. 131I appeared in bile a little more than125I and 125I/131I ratio was 0.86.Paperelectrophoresis proved that 93-97% of the radioactivity was organic iodide.
Iodide compounds in the butanol extract were analyzed with two dimensional paper-chromatography. To simplify the identification, a method of determing the ratio of numbers of indine atoms of 3', 5'positions and those of 3, 5 positions from the ratio of 125I and 131I of the spot on the paper was devised. With this method, the existence of the glucuronide of reverse triiodothyronine in bile suggested by Flock et al. was confirmed.
