Studies of the Peripheral Metabolism of Triiodothyronine and Reverse Triiodothyronine in the Rat: A Comparison of Extraction and Chromatographic Methods of Analysis*

Separation of Iodothyronines from Iodinated Metabolites in Serum from Reverse T3Tracer Kinetic Studies in Man and Dog:

“…These features improve the accuracy of determining small quantities of labeled iodothyronine at the end of the kinetic study, as well as allowing the use of smaller dose of labeled iodothyronines to satisfactorily perform the studies. Compared to the gel separation/antibody extraction technique (Balsam et al 1978;Faber et al 1982;Aikin et al 1977;Bianchi et al 1977;Perrild, Skovsted and Korsgaard Christensen 1980), the THF/SEP-PAK method also has the advantage of not requiring the use of specific antibodies. The latter is not necessary since very small quantities of diiodo-and monoiodothyronines have been shown to accumulate relative to reverse T 3 during the period of study (Faber et al 1982).…”

“…Other techniques for iodothyronine isolation include trichloroacetic acid (TCA)/ethanol extraction (Surks and Oppenheimer 1969) which does not effectively exclude free iodides and iodoproteins from iodothyronines (Balsam et al 1978;Faber et al 1982;Aikin, Eraser, Cooper, Hall and Burke 1977;Bianchi, Zucchelli, Giannessi, Pilo, Mariani, Carpi, Toni and Cazzuola 1977), methanol extraction followed by thin-layer chromatography (Hays et al 1980;Hays 1981 andSephadexchromatography (Sakurada et al 1978; which are laborious, and gel separation followed by antibody extraction (Balsam et al 1978;Faber et al 1982;Aikin et al 1977;Bianchi et al 1977;Perrild, Skovsted and Korsgaard Christensen 1980) which requires use of highly specific antibodies. Recently, an ammonium acetate/tetrahydrofuran (THF) elution of serum samples on C-18 SEP-PAK cartridges has been developed by LoPresti et al (Personal Communication) which is relatively simple, reproducible, allows efficient iodothyronine recovery and separation from free iodide and iodoproteins, and can be readily used for the batch-wise processing of large numbers and volumes of serum samples.…”

Peripheral Metabolism of Thyroid Hormones in Vitamin A-Deficient Rats

“…Turnover o f Thyroid Hormones 30 minutes before initiation of turnover studies, animals were given 1 mg KI by intraperitoneal injec tion to block thyroid uptake of radioiodine liberated from administered labeled hormones [14]. At 0 time, animals were given subcutaneous injections of 1 pCi (125I)-T4 (Tetra 125-B, CEA Saclay, France; specific activity = 150pCi/pg) or 5 pCi (1J5I)-Tj (IM 32, Amersham; specific activity = 50 pCi/pg) in 200 pi phosphate buffer.…”

“…In the treatment of serum containing(l25I)-T3 [14], 50 pi of serum were enriched with carrier Nal (1 mM) and methimazole (5 mM). Samples were combined with 100 pi of outdated blood-bank serum and treated with 600 pi of 20% TCA.…”

“…In the two experiments, 'TCA-precipitable, etha nol-extractable radioactivity was calculated as the dif ference between counts obtained in the precipitate before and after alcohol precipitation; this has been considered to represent radioactivity in iodothyronines, as radioiodine in iodide and iodoprotein has been essentially excluded' [14].…”