Abstract
3,5-diiodo-L-thyronine (3,5-T2) is an endogenous derivative of thyroid hormone with potential metabolic effects. It has been detected in human blood by immunological methods, but a reliable assay based on mass spectrometry (MS), which is now regarded as the gold standard in clinical chemistry, is not available yet. Therefore, we aimed at developing a novel ad-hoc optimized method to quantitate 3,5-T2 and its isomers by MS in human serum. Serum samples were obtained from 28 healthy subjects. Two ml of serum were deproteinized with acetonitrile and then subjected to an optimized solid phase extraction-based procedure. To lower background noise, the samples were furtherly cleaned by hexane washing and acetonitrile precipitation of residual proteins. 3,5-T2 and its isomers 3,3′-T2 and 3′,5′-T2 were then analyzed by HPLC coupled to tandem MS. Accuracy and precision for T2 assay were 88–104% and 95–97%, respectively. Recovery and matrix effect averaged 78% and +8%, respectively. 3,5-T2 was detected in all samples and its concentration averaged (mean ± SEM) 41 ± 5 pg/ml, i.e., 78 ± 9 pmol/l. In the same samples the concentration of 3,3′-T2 averaged 133±15 pg/ml, i.e., 253±29 pmol/l, while 3′,5′-T2 was not detected. 3,5-T2 concentration was significantly related to 3,3′-T2 concentration (r = 0.540, P < 0.01), while no significant correlation was observed with either T3 or T4 in a subset of patients in which these hormones were assayed. In conclusion, our method is able to quantify 3,5-T2 and 3,3′-T2 in human serum. Their concentrations lie in the subnanomolar range, and a significant correlation was detected between these two metabolites in healthy individuals.
Highlights
The term thyroid hormones, classically referred to both 3,5,3′-triiodothyronine (T3) and thyroxine (T4), seems nowadays to be simplistic; it has been shown that some T3 and T4 metabolites, 3,5-diiodothyronine (3,5-T2) and 3-iodothyronamine, are independent chemical messengers, with specific metabolic effects [1].Assay of Endogenous 3,5-T2 and 3,3′-T2Two deiodinase enzymes, namely D1- and D3-deiodinase, can potentially catalyse the synthesis of distinct diiodothyronines: 3,5-diiodothyronine (3,5-T2), 3,3′- diiodothyronine (3,3′-T2) and 3′,5′- diiodothyronine (3′,5′-T2) [2]
Representative chromatograms obtained in two patients are shown in Figure 1. 3,5-T2 and 3,3′-T2 were present in all samples, while 3′,5′-T2 was never detected
Due to the emerging role of 3,5-T2 in lipid and glucose metabolism, the adequate measurement of 3,5-T2 and related compound is a challenging aim of thyroid hormone research
Summary
The term thyroid hormones, classically referred to both 3,5,3′-triiodothyronine (T3) and thyroxine (T4), seems nowadays to be simplistic; it has been shown that some T3 and T4 metabolites, 3,5-diiodothyronine (3,5-T2) and 3-iodothyronamine, are independent chemical messengers, with specific metabolic effects [1].Assay of Endogenous 3,5-T2 and 3,3′-T2Two deiodinase enzymes, namely D1- and D3-deiodinase, can potentially catalyse the synthesis of distinct diiodothyronines: 3,5-diiodothyronine (3,5-T2), 3,3′- diiodothyronine (3,3′-T2) and 3′,5′- diiodothyronine (3′,5′-T2) [2]. All T2 isomers were regarded as inactive metabolites of T3 and T4, because of their very low affinity for nuclear thyroid hormone receptors. This view has been challenged by the observation that 3,5-T2 can interact with mitochondrial targets and that administration of exogenous 3,5-T2 to experimental animals produces significant functional effects on lipid metabolism and mitochondrial function [3]. In 1982 Faber et al reported a serum 3,5-T2 concentration close to 100 pmol/L with a RIA method based on gel separation and antibody extraction (GSAE) [5]. The average concentration was 430 pmol/L in 31 hypothyroid patients and 310 pmol/L in 24 hyperthyroid patients, vs. 290 pmol/L in the control healthy group, this difference was not statistically significant [7]
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