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

Abstract

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.

Translating Pharmacological Findings from Hypothyroid Rodents to Euthyroid Humans: Is There a Functional Role of Endogenous 3,5-T2?

“…For this purpose, we used a newly developed immunoassay (20), representing the only currently available method to determine serum 3,5-T2 concentrations in humans (26)(27)(28). Our data revealed significant bell-shaped associations of serum 3,5-T2 concentrations with serum glucose concentration and TSH concentrations in the fasting state, pointing toward a versatile role of circulating 3,5-T2 in human metabolism.…”

American Thyroid Association Guide to Investigating Thyroid Hormone Economy and Action in Rodent and Cell Models

“…A concern is the sample volume needed for free hormone determination, which is still relatively large for applications involving mice, except in terminal bleeding. Similar methodology applying liquid chromatographytandem mass spectrometry has been developed for measurement of iodothyronamines, a decarboxylated iodothyronine present in a number of biological fluids (74). & …”

Essential Molecular Determinants for Thyroid Hormone Transport and First Structural Implications for Monocarboxylate Transporter 8

“…The uptake of T 3 , T 3 AM, and TRIAC by wild-type MCT8 and the mutants R445A and D498A was directly measured by liquid chromatography-tandem mass spectrometry (LC-MS/ MS) (17,18). The cells were incubated for 3 min in DMEM-F12 (1:1) with a final concentration of 50 M. Then the medium was removed, and the cells were washed once with ice-cold PBS and harvested.…”

“…Simultaneous detection of all substances in the same biological sample was performed using a modified LC-MS/MS method as described before (18). With the 4000QTRAP triple-quadrupole tandem mass spectrometer, we reduced the time per run to 25 min using a gradient elution program: mobile phase A (water/acetonitrile/acetic acid, 95:5:0.6), mobile phase B (water/acetonitrile/acetic acid 5:95:0.6), 0 -2 min, 2% B; 2-12 min, 40% B; 12-15 min, 60% B; 15-15.5 min, 90% B; 15.5-16.5 min, 90% B; 16.5-17 min, 2% B; 17-19.95 min, 2% B.…”

“…However, this type of experiment represents an indirect approach. We took advantage of our LC-MS/MS assays for iodothyronines and TAM (17,18) and directly measured uptake of unlabeled ligands into MCT8-transfected MDCK1 cells. In a first step, we validated the LC-MS/MS method in direct comparison with the established [ 125 I]T 3 uptake assay (Fig.…”