MEASUREMENTS OF 125I LABELLED METABOLITES IN HUMAN THYROID TISSUE

Abstract

ABSTRACT The results of paper-chromatographic separation and measurements of radioactive-labelled, iodinated metabolites in digested thyroid tissue depend to a great extent on the procedure chosen. The purpose of the present study was to develop a valid and reproducible procedure for human thyroid specimens. We have systematically evaluated the following steps in the procedure: 1) removal of representative tissue for mean distribution of 125I and [125I] aminoacids, 2) chromatography of undigested thyroid homogenate, 3) propylthiouracil (PTU) versus 1-methyl-2-mercaptoimidazole (MMI) in the digest buffer, 4) digestion time using Pronase, 5) digestion of »whole« versus centrifuged homogenate, 6) chromatography of »whole«-digest versus supernatant of centrifuged digest, 7) chromatography in acid and alkaline solvents. Based on this investigation the following standard procedure was chosen: Representative tissue specimens were homogenized in Tris-acetic acid buffer 0.2 m, pH 8.6 containing PTU 7 × 10−4 m, tissue concentration 40 mg/ml. »Whole«-homogenate was digested at 37°C for 24 hours with added Pronase 4 mg/ml + toluol. »Whole«-digest was applied to Whatmann 3 MM paper for approximately 10 hours of ascending chromatography. The solvent used was n-butanol-ethanol-ammonia 0.4 n (15:3:6). The standard procedure was checked by 65 duplicate estimations and found to be highly reproducible. Extensive recovery experiments with in vitro added [125I] aminoacids and Na125I revealed a total radioactivity recovery of 96% for DIT, 93% for MIT, 95% for I−, 98% for T4 and 90% for T3. The relative recovery, an expression of the stability of individual compound, was about 100% for MIT, DIT and I−, but only 75% for T4 and 77% for T3.