HPLC analysis of in vitro hepatic deiodination products of thyroid hormones in the rainbow trout, Oncorhynchus mykiss.

Reverse-phase HPLC employing five different solvent systems was used to determine the 125I-labeled products formed in rainbow trout by in vitro incubation at 12 degrees of the hepatic microsome fraction with L-thyroxine (T4) labeled with 125I in the outer phenyl ring. Trout were starved for 2 weeks or fed a 2% ration. The only labeled products identified during incubation for 7.5-70 min over a T4 substrate range of 0.03-0.5 nM were 125I- and 3,5,[125I]3'-triiodo-L-thyronine (T3). These products in combination with the parent [125I]T4 accounted for over 96% of the total chromatographic radioactivity. Neither 3,[125I]3'-diiodo-L-thyronine nor 3,[125I]3',5'-T3 (reverse T3) was detected, suggesting negligible inner-ring deiodination of T4 or T3. Essentially equal production of 125I- and [125I]T3 validated the use of 125I- production as a measure of [125I]T3 generation in assays for hepatic 5'-monodeiodinase activity. However, in some experiments the 125I- level slightly exceeded the [125I]T3 level, indicating that outer-ring deiodination of T3 may occur to a limited degree. In conclusion, the present data for liver support earlier observations from in vivo studies in showing that for trout at 12 degrees deiodination pathways are geared primarily toward T4 outer-ring monodeiodination to produce T3 with undetectable inner-ring deiodination of T3 or T4 and limited outer-ring deiodination of T3.