A method for the determination of type I iodothyronine deiodinase activity in liver and kidney using 125I-labelled reverse triiodothyronine as a substrate

Objectives: A detailed method for the determination of iodothyronine deiodinase type I (DI-I) activity is described. The objective of the present method development was to consolidate the effective procedures of previous methods and produce an efficient assay that can be easily reproduced. Design and methods: This method uses a 5′,-125I-labelled rT3 as substrate and ion-exchange chromatography to separate released ionic iodine. Released 125I- collected in the eluate is counted, and the results used to calculate DI-I activity. Results: Results were found to be linear for tissue homogenates containing 3–11 mg protein • mL−1. Day-to-day coefficient of variation of liver homogenate was determined to be 13%. Conclusions: This method was found to be reliable, reproducible, and sample sizes as small as 10 μL could be readily assayed. The use of centrifuge filter units to contain the ion-exchange medium decreased handling of the material, and potential sources of error.