Does the aromatic L-amino acid decarboxylase contribute to thyronamine biosynthesis?

Thyronamines (TAM), recently described endogenous signaling molecules, exert metabolic and pharmacological actions partly opposing those of the thyromimetic hormone T(3). TAM biosynthesis from thyroid hormone (TH) precursors requires decarboxylation of the L-alanine side chain and several deiodination steps to convert e.g. L-thyroxine (T(4)) into the most potent 3-T(1)AM. Aromatic L-amino acid decarboxylase (AADC) was proposed to mediate TAM biosynthesis via decarboxylation of TH. This hypothesis was tested by incubating recombinant human AADC, which actively catalyzes dopamine production from DOPA, with several TH. Under all reaction conditions tested, AADC failed to catalyze TH decarboxylation, thus challenging the initial hypothesis. These in vitro observations are supported by detection of 3-T(1)AM in plasma of patients with AADC-deficiency at levels (46 ± 18 nM, n=4) similar to those of healthy controls. Therefore, we propose that the enzymatic decarboxylation needed to form TAM from TH is catalyzed by another unique, perhaps TH-specific, decarboxylase.

Development of a validated liquid chromatography/tandem mass spectrometry method for the distinction of thyronine and thyronamine constitutional isomers and for the identification of new deiodinase substrates.

Thyronines (THs) and thyronamines (TAMs) are two groups of endogenous iodine-containing signaling molecules whose representatives differ from each other only regarding the number and/or the position of the iodine atoms. Both groups of compounds are substrates of three deiodinase isozymes, which catalyze the sequential reductive removal of iodine from the respective precursor molecule. In this study, a novel analytical method applying liquid chromatography/tandem mass spectrometry (LC-MS/MS) was developed. This method permitted the unequivocal, simultaneous identification and quantification of all THs and TAMs in the same biological sample. Furthermore, a liquid-liquid extraction procedure permitting the concurrent isolation of all THs and TAMs from biological matrices, namely deiodinase (Dio) reaction mixtures, was established. Method validation experiments with extracted TH and TAM analytes demonstrated that the method was selective, devoid of matrix effects, sensitive, linear over a wide range of analyte concentrations and robust in terms of reproducible recoveries, process efficiencies as well as intra-assay and inter-assay stability parameters. The method was applied to study the deiodination reactions of iodinated THs catalyzed by the three deiodinase isozymes. With the HPLC protocol developed herein, sufficient chromatographic separation of all constitutional TH and TAM isomers was achieved. Accordingly, the position of each iodine atom removed from a TH substrate in a Dio-catalyzed reaction was backtracked unequivocally. While several established deiodination reactions were verified, two as yet unknown reactions, namely the phenolic ring deiodination of 3',5'-diiodothyronine (3',5'-T2) by Dio2 and the tyrosyl ring deiodination of 3-monoiodothyronine (3-T1) by Dio3, were newly identified.