Isolation of labeled triiodothyronine from serum using affinity chromatography: application to the extimation of the...

A method for the isolation of small quantities of labeled 3,5,3' -triiodothyronine (T3) from serum or thyroid extracts is described. Conjugates of rabbit anti-T3 antibody to Sepharose 4B are incubated with 0.5 to 1 ml of human or rat serum at pH 8.6 for 1 hr. The tubes are centrifuged and washed with buffer followed by 6 M guanidine to remove nonspecifically bound labeled thyroxine (T4). The fraction of T3 and T4 bound to the Sepharose conjugate varies depending on the concentration of serum in the initial incubation tubes, the T3 and T4 content, and the specificity of the antiserum used. In a system that contains 0.5 ml of normal human serum, 1 ml of glycine-acetate buffer (pH 8.6), and 0.25 ml settled Sepharose-anti-T3 conjugate, the T3 to T4 binding ratio was generally 150-200, with up to as much as 50% of T3 bound to the pellet. The coefficient of variation of the method is less than 5%, and it may be performed in a matter of hours. There is no detectable conversion of T4 to T3 during the separation process. Using this technique, conversion of T4 to T3 was evaluated in euthyroid rats after injection of 125l-T4. Over the period of 36-72 hr after injection, a ratio of T3 to T4 of 0.74 +- 0.06 x 10-2 (mean +- SE) was present in the plasma. Using the calculated metabolic clearance rates for T3 and T4 in these animals, fractional conversion of T4 to T3 was estimated to be 27%, in good agreement with results obtained by other techniques. This method would appear to be of value for specific isolation of the small quantities of T3 produced from T4 after in vivo or in vitro T4 to T3 conversion.