Comparison of sample preparation strategies for target analysis of total thyroid hormones levels in serum by liquid chromatography-quadrupole time-of-flight-mass spectrometry.

This paper describes a novel method based on liquid chromatography quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS) for target analysis of total THs in serum. Several sample preparation strategies have been evaluated to reduce matrix effect (namely, HybridSPE cartridges, supported liquid extraction, SLE and solid phase extraction, SPE). Deproteinization and further clean-up with mixed-mode SPE was selected as the best strategy for sample preparation, since achieved the cleanest extracts and reduced ionization suppression effects (between -11 and -24%). Method validation was performed by the analysis of control human serum samples. Criteria for confirming THs identity in serum extracts were based on retention times, accurate masses, isotopic pattern and MS/MS fragmentation pattern. Moreover, the quantitation capabilities of the LC-QTOF-MS method were also evaluated in terms of linearity, precision, accuracy and sensitivity by the application of matrix-matched calibration. Additionally, the developed LC-QTOF-MS method successfully provides qualitative information on endogenous components responsible of ion suppression (e.g. lysophosphatidylcholines), via post acquisition data analysis. This demonstrates the significant advantage of using LC-QTOF-MS, as it allows retrospective querying of the acquired data without the need of re-injecting/re-processing the samples.

Identification of Arsenic Direct-Binding Proteins in Acute Promyelocytic Leukaemia Cells.

The identification of arsenic direct-binding proteins is essential for determining the mechanism by which arsenic trioxide achieves its chemotherapeutic effects. At least two cysteines close together in the amino acid sequence are crucial to the binding of arsenic and essential to the identification of arsenic-binding proteins. In the present study, arsenic binding proteins were pulled down with streptavidin and identified using a liquid chromatograph-mass spectrometer (LC-MS/MS). More than 40 arsenic-binding proteins were separated, and redox-related proteins, glutathione S-transferase P1 (GSTP1), heat shock 70 kDa protein 9 (HSPA9) and pyruvate kinase M2 (PKM2), were further studied using binding assays in vitro. Notably, PKM2 has a high affinity for arsenic. In contrast to PKM2, GSTP1and HSPA9 did not combine with arsenic directly in vitro. These observations suggest that arsenic-mediated acute promyelocytic leukaemia (APL) suppressive effects involve PKM2. In summary, we identified several arsenic binding proteins in APL cells and investigated the therapeutic mechanisms of arsenic trioxide for APL. Further investigation into specific signal pathways by which PKM2 mediates APL developments may lead to a better understanding of arsenic effects on APL.

Ion pair hollow fiber liquid-liquid-liquid microextraction combined with capillary electrophoresis-ultraviolet detection for the determination of thyroid hormones in human serum.

In this study, a novel, inexpensive, sensitive and selective analytical method that combines ion pair hollow fiber liquid-liquid-liquid microextraction (IP-HF-LLLME) with capillary electrophoresis-ultraviolet detection (CE-UV) was developed for the simultaneous determination of six thyroid hormones (including diiodothyronine (T2), 3,3,5-triiodo-l-thyronine (T3), 3,5,3,5-tetraiodolthyronine (T4), 3,3,5-triiodothyronine (rT3), monoiodotyrosine (MIT) and diiodotyrosine (DIT)) in human serum samples. By the addition of a low concentration of sodium dodecyl sulfate (SDS) into the donor phase as an ion pair reagent, octanol as the organic extraction solvent and 30 mmol/L Na2CO3 as acceptor phase, six analytes with different polarity and water solubility were successfully extracted simultaneously using HF-LLLME. To the best of our knowledge, this is the first time that a liquid phase microextraction technique was proposed for the extraction of thyroid hormones in real samples. The CE separations were investigated in detail. When 20 kV of voltage was applied, the six compounds were separated within 13 min in 25 mmol/L phosphate buffer (pH 2.15) containing 10% (v/v) acetonitrile and 0.5% (m/v) polyethylene glycol (PEG). Under the optimized conditions, enrichment factors (EFs) ranging from 183- to 366-fold were obtained and the limits of detection (at a signal-to-noise ratio of 3) were at sub µg/L level. The established IP-HF-LLLME-CE-UV method was successfully applied to simultaneous determination of thyroid hormones and relative compounds in human serum samples with good recoveries for the spiked samples.

Can thyroid hormone mimics affect thyroid hormone measurement by immunoassay?

OBJECTIVES: This study aims to determine whether the environmental pollutant and thyroid mimic, tetrabromobisphenol A (TBBPA), interferes with thyroid hormone measurement by immunoassays. DESIGN & METHODS: Hormone-relevant concentrations of TBBPA were added to thyroid hormone-stripped human serum and subjected to 6 different thyroid hormone immunoassays. RESULTS: TBBPA was negative in all of the thyroid hormone immunoassays tested except at very high concentration (above that expected in serum of TBBPA-exposed workers) where it gave a marginally positive result in one immunoassay (in house T4 radioimmunoassay (RIA)). CONCLUSIONS: Serum TBBPA present as a result of workplace exposure or its use as a fabric flame retardant is very unlikely to give false positive results in thyroid hormone immunoassays.

Identification of thyroid hormone response elements in vivo using mice expressing a tagged thyroid hormone receptor α1.

TRα1 (thyroid hormone receptor α1) is well recognized for its importance in brain development. However, due to the difficulties in predicting TREs (thyroid hormone response elements) in silico and the lack of suitable antibodies against TRα1 for ChIP (chromatin immunoprecipitation), only a few direct TRα1 target genes have been identified in the brain. Here we demonstrate that mice expressing a TRα1-GFP (green fluorescent protein) fusion protein from the endogenous TRα locus provide a valuable animal model to identify TRα1 target genes. To this end, we analysed DNA-TRα1 interactions in vivo using ChIP with an anti-GFP antibody. We validated our system using established TREs from neurogranin and hairless, and by verifying additional TREs from known TRα1 target genes in brain and heart. Moreover, our model system enabled the identification of novel TRα1 target genes such as RNF166 (ring finger protein 166). Our results demonstrate that transgenic mice expressing a tagged nuclear receptor constitute a feasible approach to study receptor-DNA interactions in vivo, circumventing the need for specific antibodies. Models like the TRα1-GFP mice may thus pave the way for genome-wide mapping of nuclear receptor-binding sites, and advance the identification of novel target genes in vivo.

Morphology and efficiency of poly(styrene-co-divinylbenzene)-based monolithic capillary columns for the separation of small and large molecules.

The morphology of organic monolithic stationary phases based on poly(styrene-divinylbenzene) was modified by changing the ratio of monomers to microporogen in order to make them also suitable for small molecule separations. The morphology of the columns was characterized by high-resolution scanning electron micrography, showing larger primary globules and larger macropores, as well as no mesopores >20 nm in the monolithic skeleton. The permeability of the modified monoliths was approximately three times higher than that of columns which have been optimized for large molecule separations, enabling operation of a 30 cm long column at pressures below 250 bar. In the isocratic separation of dansylated amino acids, plate counts of 50000-107000 m(-1) were achievable, which are equivalent to efficiencies obtained with 3.1 µm porous particles. The separation performance for small molecules in gradient elution was investigated using mixtures of dansylated amino acids, ß-lactam antibiotics, and thyroid hormones. Finally, the modified monolithic capillary columns also proved to be highly efficient in the separation of biopolymers such as peptides and proteins, enabling peak width at half height of 3-8 s and peak capacities of 110-180 in 15-30 min gradient runs.

Selective separation of hydroxy polychlorinated biphenyls (HO-PCBs) by the structural recognition on the molecularly imprinted polymers: direct separation of the thyroid hormone active analogues from mixtures.

We developed novel separation media for hydroxy polychlorinated biphenyls (HO-PCBs) using the molecular imprinting techniques. The results of evaluation for the molecularly imprinted polymers (MIPs) by the liquid chromatography (LC) suggested that MIPs had selective separation ability for certain HO-PCB analogues. The results of the LC evaluations and molecular modeling indicated that the molecular volumes and pK(a) values of template molecules were related with the retention factor of HO-PCBs. Additionally, according to the detail evaluation toward the selective separation behaviors of MIPs, these HO-PCB analogues have low pK(a) values dependent on their chemical structures. In other words, the prepared MIPs had selective recognition ability against the analogues, which have an OH group on a phenyl carbon and two chlorine atoms on the both neighboring carbons of the carbon attached with the OH group. Moreover, these analogues may have a potential for thyroid hormone activities so that we attempted to separate these analogues directly from mixtures of HO-PCBs using a prepared MIP.

Synthesis of thyroid hormone binding proteins transthyretin and albumin by human trophoblast.

CONTEXT: Mechanisms regulating materno-fetal transfer of thyroid hormone are not well understood. Modulation of trophoblast type 3 iodothyronine deiodinase (D3) may play an important role. OBJECTIVE: The objective of this study was to investigate trophoblast thyroid hormone binding proteins that may modulate interactions between D3 and T4. DESIGN: Placentas were obtained by informed consent from women delivering normal infants by repeat cesarean section at 38-40 wk gestation. T4 and T3 binding was examined in human placenta. Serum thyroid hormone binding proteins were identified by Western blotting, and their mRNA was examined by RT-PCR. Presence of these proteins in trophoblast was determined by immunocytochemistry and immunofluorescence. Cytosol was progressively purified to reveal additional thyroid hormone binding proteins that were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Effects of mefenamic acid on placental deiodination were examined by HPLC. RESULTS: We detected high-affinity T4 and T3 binding in human placental cytosol. All three major serum-binding proteins, T4 binding globulin (TBG), transthyretin (TTR), and albumin, were present in cytosol. TTR mRNA and albumin mRNA were detected in human placenta, and TTR and albumin were identified histochemically in syncytiotrophoblasts. Neither TBG mRNA nor TBG was detected, suggesting that plasma TBG had contaminated the cytosol preparation. Low-affinity thyroid hormone binding proteins alpha-1-antitrypsin and alpha-1-acid glycoprotein were also identified. Addition of mefenamic acid, a potent inhibitor of thyroid hormone binding, to placental cytosol significantly enhanced deiodination of T4 by D3. CONCLUSIONS: Placenta produces a series of thyroid hormone binding proteins that may modify thyroid hormone deiodination and materno-fetal thyroid hormone transport.

An endogenous signal triggering erythroid differentiation: identification as thyroid hormone.

The identification of thyroid hormone as an endogenous signal for erythroid differentiation began with our studies on the spontaneously differentiating murine erythroleukemia clone 3-1. We observed that the spontaneous differentiation frequency was dependent on a heat stable factor present in fetal calf serum or calf bone marrow. We also noted that the bone marrow extract stimulated erythroid colony-forming units in mouse bone marrow cells, suggesting the relevance of this factor in normal erythroid differentiation. The bone marrow extract did not supplant the requirement of erythropoietin but was synergistic. Purification of the bone marrow extract indicated that the differentiation-inducing activity for clone 3-1 cells cochromatographed with a low-molecular-weight, UV (280 nm)-absorbing component(s). These observations and previous reports identifying the avian erythroblastosis virus oncogene v-erbA as a mutated thyroid hormone receptor which blocked erythroid differentiation led us to test thyroid hormone in our assay. Both triiodothyronine and thyroxine were highly active, and the active constituents in the chromatographically purified fraction were identified as triiodothyronine and thyroxine. Although thyroid hormone action has been associated with both in vivo and in vitro erythroid differentiation, its role has been often relegated to a secondary status. We suggest that thyroid hormone is required for the commitment of erythroid cells to terminal differentiation.

[Thyroid diagnosis 1993]. / Schilddrüsendiagnostik 1993.

Functional disorders of the thyroid and thyroidal tumors are a common challenge in daily practice. TSH of the 2nd and the 3rd generation stand in first place of the biochemical parameters in use. The FT4-test serves as confirming investigation. Additional investigations are reserved for special indications. Fine-needle aspiration performed by an experienced endocrinologist has the highest value to determine the benign character of a tumor of the thyroid. Anatomy can be optimally visualized by the modern ultrasound techniques. The most important indication for scintigraphy today is a scheduled therapy with radioactive iodine.

Iodothyronine 5-deiodinase in rat posterior pituitary.

We first describe the presence of iodothyronine 5-deiodinase (5D) in the neural lobe of rat pituitary. 6-n-Propyl-2-thiouracil (PTU), a specific inhibitor of type-I deiodinase, had no effect, showing that 5D in neurohypophysis is of type-III isozyme, which is specific for 5-deiodination and has been found only in the brain, placenta and skin. The presence of 5D (type-III) together with our previous report of 5'-deiodinase (type-I in euthyroidism and type-II in hypothyroidism) shows that the isozymes of deiodinases in the neurohypophysis are quite similar to those in the brain. These data suggest a previously unrecognized role of thyroid hormone in posterior pituitary physiology.

Thyroid hormone extraction by plasma exchange: a study of extraction rate.

How to obtain an optimal efficiency of plasma exchanges in the treatment of severe hyperthyroidism has not been defined. In order to evaluate how long the exchanges must be continued to be fully effective in extracting thyroid hormones, we evaluated the extraction rate by repeated plasma sampling in two hyperthyroid patients and three euthyroid subjects who underwent a total of seven exchanges. Plasma concentrations of thyroid hormones were also determined just before, just after, and 24 hours following the exchange. The hormonal removal rate did not fall dramatically during the exchange, so that its efficiency--in terms of hormone extraction--depends closely on its duration. The determination of plasma thyroid hormone concentrations after the exchange does not appear to be useful in evaluating the thyroid hormone loss since these concentrations may not change in spite of the hormonal extraction.

A simple method for the separation and quantitation of radiolabeled thyroid hormones in thyroxine clearance studies.

A method was developed to facilitate the separation and quantitation of radiolabeled thyroxine in plasma for thyroxine clearance studies. Following intravenous injection of radioactive thyroxine, the radiolabeled thyroid hormones were isolated from plasma protein and polar metabolites by solid phase extraction on a C18 sorbent bed. The individual thyroid hormones were then separated by ion-pair reversed phase chromatography and sequentially eluted through a UV detector and radiochromatographic detector. The radioactivity of individual radiolabeled thyroid hormones was corrected for recovery of carrier as determined from UV absorbance. The recoveries of thyroxine and 3,5,3'-triiodothyronine (T3) were 96% and 101%, respectively.

High-performance liquid chromatographic separation of iodoamino acids for tracer turnover studies of thyroid hormones in vivo.

A reversed-phase high-performance liquid chromatographic technique was developed to separate radioiodinated thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3) and two diiodothyronines (3,3'-T2 and 3',5'-T2), in extracts from either serum or urine. Chromatography was performed with 10-micron C18 silica gel, packed in a glass column (3 X 300 mm); the mobile phase was methanol-water (55:45) adjusted to pH 3 with H3PO4, at a flow-rate of 1.2 ml/min and a pressure of 2800 p.s.i. The results demonstrate the ability of the system to yield a clear-cut separation of the iodothyronines involved in in vivo turnover studies, i.e., T4, T3, rT3, and the two T2 compounds together.

Passage of thyroid hormone into milk in rabbits.

In lactating rabbits of New-Zealand breed the passage of iodothyronines from blood to milk was studied. The iodothyronines from the milk were extracted with different solvent systems and then separated either with the aid of paper chromatography in butanol-ethanol-ammonia system or on a Sephadex G-25 fine columns with the use of alkalized ethanol. At 4 h after the administration of labelled compounds the milk/serum ratio for thyroxine (T4) was 0.3, for triiodothyronine (T3) 1.2 to 2.8 and for reverse triiodothyronine (rT3) 9.0 to 18.0. When estimated with the aid of specific radioimmunoassay, the concentration of thyroxine was about 25%, while that of T3 was about 50% and that of rT3 was about 300% of the level of the corresponding compound found in plasma.

Isolation of radioactive iodothyronines for kinetic studies: a comparison of two methods.

A method based on the principle of gel separation followed by antibody extraction (GSAE) has been developed for isolation of radioactive thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3'5'-triiodothyronine (rT3), 3,3'-diiodothyronine (3,3'-T2), 3',5'-diiodothyronine (3',5'-T2) and 3'monoiodothyronine (3'-T1) in serum. This method was used for the estimation of the metabolic clearance rate (MCR( of the iodothyronines using the single injection, non-compartmental approach, and was compared to the conventional trichloroacetic acid precipitation/ethanol extraction (TCA-E) technique. The GSAE method excluded the co-determination of radioactive iodine ad iodoproteins, whereas the co-determination of radiolabelled daughter iodothyronines was found negligible. The relative difference of duplicate estimation of MCR was approximately 10%. Using the TCA-E method for isolation of tracer, the MCR of T4, T3 and rT3 was underestimated to a minor degree (20%), whereas the MCRs of 3,3'-T2, 3'5'-T2 and 3'-T1 were 20-40% of the estimated by the GSAE method. In conclusion the GSAE method was found suitable for kinetic studies of iodothyronines, whereas the TCA-E method cannot be used for turnover studies of 3,3'-T2, 3'5'-T2 or 3'T1.