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

Abstract

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° of the hepatic microsome fraction with l-thyroxine (T 4) 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 T 4 substrate range of 0.03–0.5 n M were 125I − and 3,5,[ 125I]3′-triiodo- l-thyronine (T 3). These products in combination with the parent [ 125I]T 4 accounted for over 96% of the total chromatographic radioactivity. Neither 3,[ 125I]3′-diiodo- l-thyronine nor 3,[ 125I]3′,5′-T 3 (reverse T 3) was detected, suggesting negligible inner-ring deiodination of T 4 or T 3. Essentially equal production of 125I − and [ 125I]T 3 validated the use of 125I − production as a measure of [ 125I]T 3 generation in assays for hepatic 5′-monodeiodinase activity. However, in some experiments the 125I − level slightly exceeded the [ 125I]T 3 level, indicating that outer-ring deiodination of T 3 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° deiodination pathways are geared primarily toward T 4 outer-ring monodeiodination to produce T 3 with undetectable inner-ring deiodination of T 3 or T 4 and limited outer-ring deiodination of T 3.