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Queuosine is one of the most complex hypermodified RNA nucleosides found in the Wobble position of tRNAs. In addition to Queuosine itself, several further modified derivatives are known, where the cyclopentene ring structure is additionally modified by a galactosyl-, a mannosyl-, or a glutamyl-residue. While sugar-modified Queuosine derivatives are found in the tRNAs of vertebrates, glutamylated Queuosine (gluQ) is only known in bacteria. The exact structure of gluQ, particularly with respect to how and where the glutamyl side chain is connected to the Queuosine cyclopentene side chain, is unknown. Here we report the first synthesis of gluQ and, using UHPLC-MS-coinjection and NMR studies, we show that the isolated natural gluQ is the α-allyl-connected gluQ compound.
Assuntos
Nucleosídeo Q , RNA de Transferência , Animais , Nucleosídeo Q/química , RNA de Transferência/química , Bactérias , CiclopentanosRESUMO
The importance of thyroid hormones in the regulation of development, growth, and energy metabolism is well known. Over the last decades, mass spectrometry has been extensively used to investigate thyroid hormone metabolism and to discover and characterize new molecules involved in thyroid hormones production, such as thyrotropin-releasing hormone. In the earlier period, the quantification methods, usually based on gas chromatography-mass spectrometry, were complicated and time consuming. They were mainly focused on basic research, and were not suitable for clinical diagnostics on a routine basis. The development of the modern mass spectrometers, mainly coupled to liquid chromatography, enabled simpler sample preparation procedures, and the accurate quantification of thyroid hormones, of their precursors, and of their metabolites in biological fluids, tissues, and cells became feasible. Nowadays, molecules of physiological and pathological interest can be assayed also for diagnostic purposes on a routine basis, and mass spectrometry is slowly entering the clinical laboratory. This review takes stock of the advancements in the field of thyroid metabolism that were carried out with mass spectrometry, with special focus on the use of this technique for the quantification of molecules involved in thyroid diseases.
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Doenças da Glândula Tireoide , Hormônios Tireóideos , Cromatografia Líquida/métodos , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Espectrometria de Massas/métodos , Doenças da Glândula Tireoide/diagnóstico , Hormônios Tireóideos/análise , Hormônios Tireóideos/química , Hormônios Tireóideos/metabolismoRESUMO
Abnormalities in thyroid hormones (TH) availability and/or metabolism have been hypothesized to contribute to Alzheimer's disease (AD) and to be a risk factor for stroke. Recently, 3-iodothyronamine (T1AM), an endogenous amine putatively derived from TH metabolism, gained interest for its ability to promote learning and memory in the mouse. Moreover, T1AM has been demonstrated to rescue the ß-Amyloid dependent LTP impairment in the entorhinal cortex (EC), a brain area crucially involved in learning and memory and early affected during AD. In the present work, we have investigated the effect of T1AM on ischemia-induced EC synaptic dysfunction. In EC brain slices exposed to oxygen-glucose deprivation (OGD), we demonstrated that the acute perfusion of T1AM (5 µM) was capable of preventing ischemia-induced synaptic depression and that this protective effect was mediated by the trace amine-associated receptor 1 (TAAR1). Moreover, we demonstrated that activation of the BDNF-TrkB signalling is required for T1AM action during ischemia. The protective effect of T1AM was more evident when using EC slices from transgenic mutant human APP (mhAPP mice) that are more vulnerable to the effect of OGD. Our results confirm that the TH derivative T1AM can rescue synaptic function after transient ischemia, an effect that was also observed in a Aß-enriched environment.
Assuntos
Isquemia Encefálica/patologia , Córtex Entorrinal/patologia , Receptores Acoplados a Proteínas G/metabolismo , Tironinas/farmacologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Animais , Isquemia Encefálica/metabolismo , Córtex Entorrinal/efeitos dos fármacos , Humanos , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Recent reports highlighted the significant neuroprotective effects of thyronamines (TAMs), a class of endogenous thyroid hormone derivatives. In particular, 3-iodothyronamine (T1AM) has been shown to play a pleiotropic role in neurodegeneration by modulating energy metabolism and neurological functions in mice. However, the pharmacological response to T1AM might be influenced by tissue metabolism, which is known to convert T1AM into its catabolite 3-iodothyroacetic acid (TA1). Currently, several research groups are investigating the pharmacological effects of T1AM systemic administration in the search of novel therapeutic approaches for the treatment of interlinked pathologies, such as metabolic and neurodegenerative diseases (NDDs). A critical aspect in the development of new drugs for NDDs is to know their distribution in the brain, which is fundamentally related to their ability to cross the blood-brain barrier (BBB). To this end, in the present study we used the immortalized mouse brain endothelial cell line bEnd.3 to develop an in vitro model of BBB and evaluate T1AM and TA1 permeability. Both drugs, administered at 1 µM dose, were assayed by high-performance liquid chromatography coupled to mass spectrometry. Our results indicate that T1AM is able to efficiently cross the BBB, whereas TA1 is almost completely devoid of this property.
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Encéfalo/metabolismo , Animais , Transporte Biológico/fisiologia , Barreira Hematoencefálica/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Técnicas de Cocultura/métodos , Células Endoteliais/metabolismo , Humanos , Camundongos , Doenças Neurodegenerativas/tratamento farmacológico , Fármacos Neuroprotetores/metabolismo , Permeabilidade/efeitos dos fármacos , Tironinas/metabolismoRESUMO
In the present work, we investigated the response to Cd in Leptodictyum riparium, a cosmopolitan moss (Bryophyta) that can accumulate higher amounts of metals than other plants, even angiosperms, with absence or slight apparent damage. High-performance liquid chromatography followed by electrospray ionization tandem mass spectrometry of extracts from L. riparium gametophytes, exposed to 0, 36 and 360 µM Cd for 7 days, revealed the presence of γ-glutamylcysteine (γ-EC), reduced glutathione (GSH), and traces of phytochelatins. The increase in Cd concentrations progressively augmented reactive oxygen species levels, with activation of both antioxidant (catalase and superoxide dismutase) and detoxifying (glutathione-S-transferase) enzymes. After Cd treatment, cytosolic and vacuolar localization of thiol peptides was performed by means of the fluorescent dye monochlorobimane and subsequent observation with confocal laser scanning microscopy. The cytosolic fluorescence observed with the highest Cd concentrations was also consistent with the formation of γ-EC-bimane in the cytosol, possibly catalyzed by the peptidase activity of the L. riparium phytochelatin synthase. On the whole, activation of phytochelatin synthase and glutathione-S-transferase, but minimally phytochelatin synthesis, play a role to counteract Cd toxicity in L. riparium, in this manner minimizing the cellular damage caused by the metal. This study strengthens previous investigations on the L. riparium ability to efficiently hinder metal pollution, hinting at a potential use for biomonitoring and phytoremediation purposes.
Assuntos
Aminoaciltransferases/metabolismo , Bryopsida/efeitos dos fármacos , Bryopsida/metabolismo , Cádmio/metabolismo , Cádmio/toxicidade , Glutationa Transferase/metabolismo , Fitoquelatinas/metabolismo , Estresse Fisiológico/fisiologia , Antioxidantes , Biodegradação Ambiental , Monitoramento Biológico , Cádmio/administração & dosagem , Parede Celular/metabolismo , Clorofila , Células Germinativas Vegetais , Glutationa , Metais , Espécies Reativas de Oxigênio/metabolismoRESUMO
Eukaryotic chromatin is organized into functional domains, that are characterized by distinct proteomic compositions and specific nuclear positions. In contrast to cellular organelles surrounded by lipid membranes, the composition of distinct chromatin domains is rather ill described and highly dynamic. To gain molecular insight into these domains and explore their composition, we developed an antibody-based proximity biotinylation method targeting the RNA and proteins constituents. The method that we termed antibody-mediated proximity labelling coupled to mass spectrometry (AMPL-MS) does not require the expression of fusion proteins and therefore constitutes a versatile and very sensitive method to characterize the composition of chromatin domains based on specific signature proteins or histone modifications. To demonstrate the utility of our approach we used AMPL-MS to characterize the molecular features of the chromocenter as well as the chromosome territory containing the hyperactive X chromosome in Drosophila. This analysis identified a number of known RNA-binding proteins in proximity of the hyperactive X and the centromere, supporting the accuracy of our method. In addition, it enabled us to characterize the role of RNA in the formation of these nuclear bodies. Furthermore, our method identified a new set of RNA molecules associated with the Drosophila centromere. Characterization of these novel molecules suggested the formation of R-loops in centromeres, which we validated using a novel probe for R-loops in Drosophila. Taken together, AMPL-MS improves the selectivity and specificity of proximity ligation allowing for novel discoveries of weak protein-RNA interactions in biologically diverse domains.
Assuntos
Cromatina , Espectrometria de Massas , RNA , Animais , Cromatina/metabolismo , Cromatina/química , RNA/metabolismo , RNA/química , Espectrometria de Massas/métodos , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Biotinilação , Centrômero/metabolismo , Anticorpos/metabolismo , Anticorpos/química , Cromossomo X/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/química , Coloração e Rotulagem/métodosRESUMO
OBJECTIVE: Retinitis pigmentosa (RP) is a hereditary retinal disease characterized by progressive photoreceptor degeneration, leading to vision loss. The best hope for a cure for RP lies in gene therapy. However, given that RP patients are most often diagnosed in the midst of ongoing photoreceptor degeneration, it is unknown how the retinal proteome changes as RP disease progresses, and which changes can be prevented, halted, or reversed by gene therapy. METHODS: Here, we used a Pde6b-deficient RP gene therapy mouse model and performed untargeted proteomic analysis to identify changes in protein expression during degeneration and after treatment. RESULTS: We demonstrated that Pde6b gene restoration led to a novel form of homeostatic plasticity in rod phototransduction which functionally compensates for the decreased number of rods. By profiling protein levels of metabolic genes and measuring metabolites, we observed an upregulation of proteins associated with oxidative phosphorylation in mutant and treated photoreceptors. CONCLUSION: In conclusion, the metabolic demands of the retina differ in our Pde6b-deficient RP mouse model and are not rescued by gene therapy treatment. These findings provide novel insights into features of both RP disease progression and long-term rescue with gene therapy.
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Background: Congenital hypothyroidism due to defects in iodotyrosine deiodinase has variable phenotypes and can present as hypothyroid or with normal thyroid testing. Methods: Whole exome sequencing was performed in individuals from two families originating from different regions of Sudan. Mass spectrometry of urine and serum iodotyrosines was performed on subjects from both families. Results: A novel iodotyrosine deiodinase (IYD) mutation (c.835C>T; R279C) was identified in individuals from two Sudanese families inherited as autosomal recessive. The mutation was identified by multiple in silica analyses to likely be detrimental. Serum and urine monoiodotyrosine (MIT) and diiodotyrosine (DIT) were markedly elevated in the homozygous subjects. Conclusion: Measurement of serum and urine DIT and MIT was more sensitive than that of urine iodine or serum thyroid function tests to determine the effect of the IYD mutation.
Assuntos
Hipotireoidismo Congênito , Di-Iodotirosina , Mutação , Humanos , Hipotireoidismo Congênito/genética , Di-Iodotirosina/genética , Iodeto Peroxidase/genética , Monoiodotirosina/genéticaRESUMO
Background: Iodine is required for the synthesis of thyroid hormone (TH), but its natural availability is limited. Dehalogenase1 (Dehal1) recycles iodine from mono- and diiodotyrosines (MIT, DIT) to sustain TH synthesis when iodine supplies are scarce, but its role in the dynamics of storage and conservation of iodine is unknown. Methods: Dehal1-knockout (Dehal1KO) mice were generated by gene trapping. The timing of expression and distribution was investigated by X-Gal staining and immunofluorescence using recombinant Dehal1-beta-galactosidase protein produced in fetuses and adult mice. Adult Dehal1KO and wild-type (Wt) animals were fed normal and iodine-deficient diets for 1 month, and plasma, urine, and tissues were isolated for analyses. TH status was monitored, including thyroxine, triiodothyronine, MIT, DIT, and urinary iodine concentration (UIC) using a novel liquid chromatography with tandem mass spectrometry method and the Sandell-Kolthoff (S-K) technique throughout the experimental period. Results: Dehal1 is highly expressed in the thyroid and is also present in the kidneys, liver, and, unexpectedly, the choroid plexus. In vivo transcription of Dehal1 was induced by iodine deficiency only in the thyroid tissue. Under normal iodine intake, Dehal1KO mice were euthyroid, but they showed negative iodine balance due to a continuous loss of iodotyrosines in the urine. Counterintuitively, the UIC of Dehal1KO mice is twofold higher than that of Wt mice, indicating that S-K measures both inorganic and organic iodine. Under iodine restriction, Dehal1KO mice rapidly develop profound hypothyroidism, while Wt mice remain euthyroid, suggesting reduced retention of iodine in the thyroids of Dehal1KO mice. Urinary and plasma iodotyrosines were continually elevated throughout the life cycles of Dehal1KO mice, including the neonatal period, when pups were still euthyroid. Conclusions: Plasma and urine iodotyrosine elevation occurs in Dehal1-deficient mice throughout life. Therefore, measurement of iodotyrosines predicts an eventual iodine shortage and development of hypothyroidism in the preclinical phase. The prompt establishment of hypothyroidism upon the start of iodine restriction suggests that Dehal1KO mice have low iodine reserves in their thyroid glands, pointing to defective capacity for iodine storage.
Assuntos
Hipotireoidismo , Iodo , Camundongos , Animais , Monoiodotirosina/metabolismo , Camundongos Knockout , Iodeto Peroxidase/genética , Hipotireoidismo/genética , Biomarcadores , Tiroxina , Iodo/metabolismoRESUMO
T1AM, a derivative of thyroid hormones, and its major catabolite, TA1, produce effects on memory acquisition in rodents. In the present study, we compared the effects of exogenous T1AM and TA1 on protein belonging to signal transduction pathways, assuming that TA1 may strengthen T1AM's effects in brain tissue. A hybrid line of cancer cells of mouse neuroblastoma and rat glioma (NG 108-15), as well as a human glioblastoma cell line (U-87 MG) were used. We first characterized the in vitro model by analyzing gene expression of proteins involved in the glutamatergic cascade and cellular uptake of T1AM and TA1. Then, cell viability, glucose consumption, and protein expression were assessed. Both cell lines expressed receptors implicated in glutamatergic pathway, namely Nmdar1, Glur2, and EphB2, but only U-87 MG cells expressed TAAR1. At pharmacological concentrations, T1AM was taken up and catabolized to TA1 and resulted in more cytotoxicity compared to TA1. The major effect, highlighted in both cell lines, albeit on different proteins involved in the glutamatergic signaling, was an increase in phosphorylation, exerted by T1AM but not reproduced by TA1. These findings indicate that, in our in vitro models, T1AM can affect proteins involved in the glutamatergic and other signaling pathways, but these effects are not strengthened by TA1.
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Legionella pneumophila (L.p.) is a bacterial pathogen which is a common causative agent of pneumonia. In humans, it infects alveolar macrophages and transfers hundreds of virulence factors that interfere with cellular signalling pathways and the transcriptomic landscape to sustain its own replication. By this interaction, it has acquired eukaryote-like protein motifs by gene transfer events that partake in the pathogenicity of Legionella. In a computational screening approach for eukaryotic motifs in the transcriptome of Legionella, we identified the L.p. strain Corby protein ABQ55614 as putative histone-deacetylase and named it "suppressing modifier of histones 1" (Smh1). During infection, Smh1 is translocated from the Legionella vacuole into the host cytosol. When expressed in human macrophage THP-1 cells, Smh1 was localized predominantly in the nucleus, leading to broad histone H3 and H4 deacetylation, blunted expression of a large number of genes (e.g. IL-1ß and IL-8), and fostered intracellular bacterial replication. L.p. with a Smh1 knockdown grew normally in media but showed a slight growth defect inside the host cell. Furthermore, Smh1 showed a very potent histone deacetylation activity in vitro, e.g. at H3K14, that could be inhibited by targeted mutation of the putative catalytic center inferred by analogy with eukaryotic HDAC8, and with the deacetylase inhibitor trichostatin A. In summary, Smh1 displays functional homology with class I/II type HDACs. We identified Smh1 as a new Legionella virulence factor with a eukaryote-like histone-deacetylase activity that moderates host gene expression and might pave the way for further histone modifications.IMPORTANCELegionella pneumophila (L.p.) is a prominent bacterial pathogen, which is a common causative agent of pneumonia. In order to survive inside the host cell, the human macrophage, it profoundly interacts with host cell processes to advance its own replication. In this study, we identify a bacterial factor, Smh1, with yet unknown function as a host histone deacetylase. The activity of this factor in the host cell leads to attenuated gene expression and increased intracellular bacterial replication.
Assuntos
Eucariotos , Legionella pneumophila , Humanos , Histonas/genética , Legionella pneumophila/genética , Células Eucarióticas , Pesquisa , Fatores de Virulência/genética , Histona Desacetilases , Proteínas RepressorasRESUMO
This paper reports functional studies on the enzyme phytochelatin synthase in the liverwort Marchantia polymorpha and the cyanobacterium Geitlerinema sp. strain PCC 7407. In vitro activity assays in control samples (cadmium-untreated) showed that phytochelatin synthase was constitutively expressed in both organisms. In the presence of 100 µM cadmium, in both the liverwort and the cyanobacterium, the enzyme was promptly activated in vitro, and produced phytochelatins up to the oligomer PC4. Likewise, in vivo exposure to 10-36 µM cadmium for 6-120 h induced in both organisms phytochelatin synthesis up to PC4. Furthermore, the glutathione (GSH) levels in M. polymorpha were constitutively low (compared with the average content in higher plants), but increased considerably under cadmium stress. Conversely, the GSH levels in Geitlerinema sp. PCC 7407 were constitutively high, but were halved under metal treatments. At odds with former papers, our results demonstrate that, as in M. polymorpha and other plants, the cyanobacterial phytochelatin synthase exposed to cadmium possesses manifest transpeptidasic activity, being able to synthesize phytochelatins with a degree of oligomerization higher than PC2. Therefore, prokaryotic and eukaryotic phytochelatin synthases differ less in functional terms than previously thought.
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Background: A novel form of thyroid hormone (TH) signaling is represented by 3-iodothyronamine (T1AM), an endogenous TH derivative that interacts with specific molecular targets, including trace amine-associated receptor 1 (TAAR1), and induces pro-learning and anti-amnestic effects in mice. Dysregulation of TH signaling has long been hypothesized to play a role in Alzheimer's disease (AD). In the present investigation, we explored the neuroprotective role of T1AM in beta amyloid (Aß)-induced synaptic and behavioral impairment, focusing on the entorhinal cortex (EC), an area that is affected early by AD pathology. Methods: Field potentials were evoked in EC layer II, and long-term potentiation (LTP) was elicited by high frequency stimulation (HFS). T1AM (5 µM) and/or Aß(1-42) (200 nM), were administered for 10 minutes, starting 5 minutes before HFS. Selective TAAR1 agonist RO5166017 (250 nM) and TAAR1 antagonist EPPTB (5 nM) were also used. The electrophysiological experiments were repeated in EC-slices taken from a mouse model of AD (mutant human amyloid precursor protein [mhAPP], J20 line). We also assessed the in vivo effects of T1AM on EC-dependent associative memory deficits, which were detected in mhAPP mice by behavioral evaluations based on the novel-object recognition paradigm. TAAR1 expression was determined by Western blot, whereas T1AM and its metabolite 3-iodothyroacetic acid (TA1) were assayed by high-performance liquid chromatography coupled to mass spectrometry. Results: We demonstrate the presence of endogenous T1AM and TAAR1 in the EC of wild-type and mhAPP mice. Exposure to Aß(1-42) inhibited LTP, and T1AM perfusion (at a concentration of 5 µM, leading to an actual concentration in the perfusion buffer ranging from 44 to 298 nM) restored it, whereas equimolar amounts of 3,5,3'-triiodo-L-thyronine (T3) and TA1 were ineffective. The response to T1AM was abolished by the TAAR1 antagonist EPPTB, whereas it was mimicked by the TAAR1 agonist RO5166017. In the EC of APPJ20 mice, LTP could not be elicited, but it was rescued by T1AM. The intra-cerebro-ventricular administration of T1AM (0.89 µg/kg) also restored recognition memory that was impaired in mhAPP mice. Conclusions: Our results suggest that T1AM and TAAR1 are part of an endogenous system that can be modulated to prevent synaptic and behavioral deficits associated with Aß-related toxicity.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Córtex Entorrinal/efeitos dos fármacos , Potenciais Evocados/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Tironinas/farmacologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Córtex Entorrinal/fisiologia , Potenciais Evocados/fisiologia , Camundongos , Camundongos TransgênicosRESUMO
Although thiol-peptide compounds, such as reduced glutathione (GSH), γ-glutamylcysteine (γ-EC), and phytochelatins, play fundamental roles in plants, their analytical determination and characterization is still somewhat problematic, mainly due to their high polarity and oxidation propensity. Thus, in this work a reliable and sensitive HPLC-ESI-MS-MS method was developed, in order to simultaneously assay, within 14-min instrumental runs, γ-EC, GSH, and phytochelatins up to phytochelatin 4. This analytical method was validated in shoot and root extracts of the model plant Arabidopsis thaliana (Brassicaceae) and guaranteed accurate quantification by using specific isotope labelled-internal standards for both GSH and phytochelatins, as well as standards for external calibration. Good linearities in the method performance were observed (Râ¯>â¯0.99), with a dynamic range over three orders of magnitude in thiol-peptide concentrations. In MRM mode, the detection sensitivity of the thiol-peptides was equal to approximately 16, 6, 7, 13, 10â¯fmol for γ-EC, GSH, phytochelatin 2, phytochelatin 3, and phytochelatin 4, respectively (20⯵l injection each). The reproducibility of the method was confirmed by high intra- and inter-day accuracy and precision values. The recovery rates were estimated approximately in the range of 73.8-91.0% and the matrix effect evaluation revealed that all analytes exhibited ionization suppression. The use of stable isotope-labelled analogs of the thiol-peptides as internal standards was particularly worthy of note: it offered the considerable advantage of overcoming the consequences of matrix effect and thiol-peptide loss through sample preparation, by normalizing the analyte signal during the quantification process. Thus, by validating the method's sensitivity, accuracy, precision, reproducibility, stability, recovery, and matrix effect, data reliability and robustness were ensured.
Assuntos
Arabidopsis/química , Peptídeos/análise , Compostos de Sulfidrila/análise , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
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.