Assay of Endogenous 3,5-diiodo-L-thyronine (3,5-T2) and 3,3′-diiodo-L-thyronine (3,3′-T2) in Human Serum: A Feasibility Study

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.

3,5-T2—A Janus-Faced Thyroid Hormone Metabolite Exerts Both Canonical T3-Mimetic Endocrine and Intracrine Hepatic Action

“…This tight binding not only relates to their distribution proteins in blood, cells or tissues but especially also to surfaces of plastic material required during pre-analytical sample work and subsequent MS quantification. Recently, two groups reported for the first time 3,5-T2 serum concentrations determined by LC-MS. Lorenzini et al (75), observed a mean concentration range for 3,5-T2 in human serum of 78 ± 9 pM, while our own results (25) indicated that most human sera analyzed for 3,5-T2 have lower than 5 pM 3,5-T2 concentrations, which is the lower limit of quantification in our method. The fact that Lorenzini et al probably report higher LC-MS-based 3,5-T2 concentrations is due to the contamination of the 13 C 9-15 N -3,5-T2 internal standard, with "natural" unlabeled 3,5-T2.…”

Genomic and Non-Genomic Mechanisms of Action of Thyroid Hormones and Their Catabolite 3,5-Diiodo-L-Thyronine in Mammals

“…Experiments in humans are indeed still limited, and, as discussed, it is not yet clear whether 3,5-T2 is produced in peripheral tissues at significant concentrations It is not completely understood, for example, whether blood concentrations of 3,5-T2 are related or not to intracellular concentrations of the hormone: It is possible that T3 deiodination to 3,5-T2 is controlled by tissue factors, differently regulated, for example, in a tissue-specific and/or in a metabolic state-specific manner. From this point of view, it is intriguing that no significant difference was found in serum 3,5-T2 of hypothyroid and hyperthyroid patients [202] and that no significant correlation was evidenced between serum 3,5-T2 and the main THs [203]. As mentioned, a further problem arises because of contamination by T3 of the commercial preparations of 3,5-T2, used as standards in the measurements [20,204].…”

“…An antibody-based competitive chemiluminescence immunoassay (CLIA) [202] was used to investigate serum concentration of 3,5-T2 in humans under both physiological and pathophysiological conditions; the results suggested that 3,5-T2 concentrations do not differ in hyperthyroid (0.31 ± 0.02 nm) compared to hypothyroid (0.43 ± 0.04 nm) individuals [202]. More recently, HPLC coupled to tandem mass spectrometry [203] allowed a more accurate 3,5-T2 detection in human serum (the average concentration reported was 78 ± 9 picomoles/liter) [203]. In general, significant differences still remain among different detection methods.…”

Pituitary Hyperplasia, Hormonal Changes and Prolactinoma Development in Males Exposed to Estrogens—An Insight From Translational Studies

“…3,5-T 2 is present in significant concentrations in human serum [141] and seems to have important implications in the regulation of energy metabolism, with a direct effect on mitochondria as the most likely mechanism [139,[141][142][143] ( Figure 6). So far, serum 3,5-T 2 concentrations have been associated with some pathophysiological states in humans, mainly related to glucose and lipid metabolism [144]. This metabolite exerts thyromimetic activity, suppresses TSH and stimulates GH [145][146][147], though not as strongly as T 3 [139].…”