A Mass Spectrometry-Based Panel of Nine Thyroid Hormone Metabolites in Human Serum.

BACKGROUND: While thyroxine (T4), 3,3',5-triiodothyronine (T3), and 3,3',5'-triiodothyronine (rT3) have routine methods available for evaluating patients with suspected thyroid disease, appropriate methods for the measurement of other thyroid hormone metabolites (THMs) are lacking. The effects of other iodothyronines or iodothyroacetic acids are therefore less explored. To better understand the (patho)physiological role of THMs, a robust method to measure iodothyronines and iodothyroacetic acids in serum in a single analysis is needed, including associated reference intervals. METHODS: Clinical and Laboratory Standards Institute guidelines, European Medicines Agency guidelines, and the National Institute of Standards and Technology protocol were used for the method validation and reference intervals. Reference intervals were determined in 132 healthy males and 121 healthy females. Serum samples were deproteinized with acetonitrile, followed by anion-exchange solid phase extraction and analysis with LC-MS/MS, using eight 13C6-internal standards. RESULTS: The analytical method validation was performed for all nine THMs. Reference intervals (2.5th to 97.5th percentile) were determined for L-thyronine (4.9-11.3 ng/dL), 3-monoiodothyronine (0.06 --0.41 ng/dL), 3,5-diiodothyronine (<0.13 ng/dL), 3,3'-diiodothyronine (0.25--0.77 ng/dL), T3 (66.4--129.9 ng/dL), rT3 (15.0--64.1 ng/dL), T4 (4.3--10.0 µg/dL), triac/3,3',5-triiodothyroacetic acid (not detected), and tetrac/3,3',5,5'-tetraiodothyroacetic acid (2.2--27.2 ng/dL). CONCLUSIONS: A broad dynamic concentration range exists among the nine THMs. This method should help to develop a better understanding of the clinical relevance of other THMs, as well as an understanding of thyroid hormone metabolism in health and disease.

Functional Characterization of the Novel and Specific Thyroid Hormone Transporter SLC17A4.

Background: A recent genome-wide association study identified the SLC17A4 locus associated with circulating free thyroxine (T4) concentrations. Human SLC17A4, being widely expressed in the gastrointestinal tract, was characterized as a novel triiodothyronine (T3) and T4 transporter. However, apart from the cellular uptake of T3 and T4, transporter characteristics are currently unknown. In this study, we delineated basic transporter characteristics of this novel thyroid hormone (TH) transporter. Methods: We performed a broad range of well-established TH transport studies in COS-1 cells transiently overexpressing SLC17A4. We studied cellular TH uptake in various incubation buffers, TH efflux, and the inhibitory effects of different TH metabolites and known inhibitors of other TH transporters on SLC17A4-mediated TH transport. Finally, we determined the effect of tunicamycin, a pharmacological inhibitor of N-linked glycosylation, and targeted mutations in Asn residues on SLC17A4 function. Results: SLC17A4 induced the cellular uptake of T3 and T4 by ∼4 times, and of reverse (r)T3 by 1.5 times over control cells. The uptake of T4 by SLC17A4 was Na+ and Cl- independent, stimulated by low extracellular pH, and reduced by various iodothyronines and metabolites thereof, particularly those that contain at least three iodine moieties irrespective of the presence of modification at the alanine side chain. None of the classical TH transporter inhibitors studied attenuated SLC17A4-mediated TH transport. SLC17A4 also facilitates the efflux of T3 and T4, and to a lesser extent of 3,3'-diiodothyronine (T2). Immunoblot studies on lysates of transfected cells cultured in absence or presence of tunicamycin indicated that SLC17A4 is subject to N-linked glycosylation. Complementary mutational studies identified Asn66, Asn75, and Asn90, which are located in extracellular loop 1, as primary targets. Conclusions: Our studies show that SLC17A4 facilitates the transport of T3 and T4, and less efficiently rT3 and 3,3'-T2. Further studies should reveal the physiological role of SLC17A4 in TH regulation.

In Vitro Characterization of Human, Mouse, and Zebrafish MCT8 Orthologues.

Background: Mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) cause MCT8 deficiency, characterized by severe intellectual and motor disability and abnormal serum thyroid function tests. Various Mct8 knock-out mouse models as well as mct8 knock-out and knockdown zebrafish models are used as a disease model for MCT8 deficiency. Although important for model eligibility, little is known about the functional characteristics of the MCT8 orthologues in these species. Therefore, we here compared the functional characteristics of mouse (mm) MCT8 and zebrafish (dr) Mct8 to human (hs) MCT8. Methods: We performed extensive transport studies in COS-1 and JEG-3 cells transiently transfected with hsMCT8, drMct8, and mmMCT8. Protein expression levels and subcellular localization were assessed by immunoblotting, surface biotinylation, and immunocytochemistry. Sequence alignment and structural modeling were used to interpret functional differences between the orthologues. Results: hsMCT8, drMct8, and mmMCT8 all facilitated the uptake and efflux of 3,3'-diiodothyronine (3,3'-T2), rT3, triiodothyronine (T3), and thyroxine (T4), although the initial uptake rates of drMct8 were 1.5-4.0-fold higher than for hsMCT8 and mmMCT8. drMct8 exhibited 3-50-fold lower apparent IC50 values than hsMCT8 and mmMCT8 for all tested substrates, and substrate preference of drMct8 (3,3'-T2, T3 > T4 > rT3) differed from hsMCT8 and mmMCT8 (T3 > T4 > rT3, 3,3'-T2). Compared with hsMCT8 and mmMCT8, cis-inhibition studies showed that T3 uptake by drMct8 was inhibited at a lower concentration and by a broader spectrum of TH metabolites. Total and cell surface expression levels of drMct8 and hsMCT8 were equal and both significantly exceeded those of mmMCT8. Structural modeling located most non-conserved residues outside the substrate pore, except for H192 in hsMCT8, which is replaced by a glutamine in drMct8. However, a H192Q substituent of hsMCT8 did not alter its transporter characteristics. Conclusion: Our studies substantiate the eligibility of mice and zebrafish models for human MCT8 deficiency. However, differences in the intrinsic transporter properties of MCT8 orthologues may exist, which should be realized when comparing MCT8 deficiency in different in vivo models. Moreover, our findings may indicate that the protein domains outside the substrate channel may play a role in substrate selection and protein stability.

Sorafenib-Induced Changes in Thyroid Hormone Levels in Patients Treated for Hepatocellular Carcinoma.

Context: The pathogenesis of tyrosine kinase inhibitor-induced thyroid hormone (TH) alterations are still a matter of debate. Objective: The objective of this study was to determine the effects of sorafenib on TH levels in patients with hepatocellular carcinoma (HCC) and to evaluate possible mechanisms. Design: We performed a prospective cohort study between 2009 and 2016. Setting: This study was conducted at a tertiary referral center. Patients: This study included 57 consecutive patients with HCC who were treated with sorafenib. Main Outcome Measure: Thyroid-stimulating hormone (TSH) and free thyroxine (FT4) levels were measured every 6 weeks, and extensive thyroid function tests (TFTs) were measured before treatment (t0), after 6 weeks (t6), and at the end of therapy. The effect of sorafenib on TH transport by monocarboxylate transporter (MCT)8 or MCT10 was tested in transfected COS1 cells. Results: Four patients (7%) developed thyroiditis. Among the other patients, 30% had elevation of TSH or FT4 above the normal range. Overall, between t0 and t6, mean TSH increased from 1.28 to 1.57 mU/L (P < 0.001) and mean FT4 from 18.4 to 21.2 pmol/L (P < 0.001). Simultaneously, the serum triiodothyronine (T3)/reverse triiodothyronine ratio and the (T3/thyroxine) ×100 ratio decreased. Sorafenib decreased cellular T3 uptake by MCT8 and to a lesser extent by MCT10. Conclusions: These in vivo data suggest that sorafenib affects TFTs on multiple levels. Our in vitro experiments suggest a possible role of sorafenib-induced inhibition of T3 transport into the cell by MCT8 and MCT10.

Tissue-Specific Suppression of Thyroid Hormone Signaling in Various Mouse Models of Aging.

DNA damage contributes to the process of aging, as underscored by premature aging syndromes caused by defective DNA repair. Thyroid state changes during aging, but underlying mechanisms remain elusive. Since thyroid hormone (TH) is a key regulator of metabolism, changes in TH signaling have widespread effects. Here, we reveal a significant common transcriptomic signature in livers from hypothyroid mice, DNA repair-deficient mice with severe (Csbm/m/Xpa-/-) or intermediate (Ercc1-/Δ-7) progeria and naturally aged mice. A strong induction of TH-inactivating deiodinase D3 and decrease of TH-activating D1 activities are observed in Csbm/m/Xpa-/- livers. Similar findings are noticed in Ercc1-/Δ-7, in naturally aged animals and in wild-type mice exposed to a chronic subtoxic dose of DNA-damaging agents. In contrast, TH signaling in muscle, heart and brain appears unaltered. These data show a strong suppression of TH signaling in specific peripheral organs in premature and normal aging, probably lowering metabolism, while other tissues appear to preserve metabolism. D3-mediated TH inactivation is unexpected, given its expression mainly in fetal tissues. Our studies highlight the importance of DNA damage as the underlying mechanism of changes in thyroid state. Tissue-specific regulation of deiodinase activities, ensuring diminished TH signaling, may contribute importantly to the protective metabolic response in aging.

In vitro glucocorticoid sensitivity is associated with clinical glucocorticoid therapy outcome in rheumatoid arthritis.

INTRODUCTION: Genetic and disease-related factors give rise to a wide spectrum of glucocorticoid (GC) sensitivity in rheumatoid arthritis (RA). In clinical practice, GC treatment is not adapted to these differences in GC sensitivity. In vitro assessment of GC sensitivity before the start of therapy could allow more individualized GC therapy. The aim of the study was to investigate the association between in vitro and in vivo GC sensitivity in RA. METHODS: Thirty-eight early and 37 established RA patients were prospectively studied. In vitro GC sensitivity was assessed with dexamethasone-induced effects on interleukin-2 (IL-2) and glucocorticoid-induced leucine zipper (GILZ) messenger RNA expression in peripheral blood mononuclear cells (PBMCs). A whole-cell dexamethasone-binding assay was used to measure number and affinity (1/KD) of glucocorticoid receptors (GRs). RESULTS: GR number was positively correlated with improvement in DAS. IL-2-EC50 and GILZ-EC50 values both had weak near-significant correlations with clinical improvement in DAS in intramuscularly treated patients only. HAQ responders had lower GILZ-EC50 values and higher GR number and KD. CONCLUSIONS: Baseline cellular in vitro glucocorticoid sensitivity is modestly associated with in vivo improvement in DAS and HAQ-DI score after GC bridging therapy in RA. Further studies are needed to evaluate whether in vitro GC sensitivity may support the development of tailor-made GC therapy in RA.