Environmental photochemical fate and UVC degradation of sodium levothyroxine in aqueous medium.

The synthetic hormone sodium levothyroxine (LTX) is one of the most prescribed drugs in the world and the most effective in hypothyroidism treatment. The presence of LTX in the environment has become a matter of major concern due to the widespread use of this hormone and by the fact that it is only partially removed in conventional water and sewage treatment plants. However, information regarding the photochemical fate of this hormone in environmental or engineered systems is scarce in the literature. In this work, the sunlight-driven direct and indirect LTX degradation was investigated by determining the photolysis quantum yield, ΦLTX = 3.80 (± 0.02) × 10-5, as well as the second-order kinetic constants of the reactions with hydroxyl radicals, kLTX,•OH = 1.50 (± 0.01) × 1010 L mol-1 s-1 and singlet oxygen, kLTX,1O2 = 1.47 (± 0.66) × 108 L mol-1 s-1. Mathematical simulations indicate that LTX photodegradation is favored in shallow, nitrite-rich, and dissolved organic matter (DOM)-poor environments, with LTX half-life times varying from less than 10 days to about 80 days. LTX removals of 85 and 95% were achieved by UVC photolysis and UVC/H2O2 after 120 min, respectively. Three transformation products, triiodothyronine, diiodothyronine, and diiodotyrosine, were identified during LTX degradation by the UVC-based processes studied. The results herein regarding photo-induced kinetics coupled with environmental fate simulations may help evaluate LTX persistence and also the design of water and wastewater treatment processes.

Effects of 3,5-Diiodotyrosine and Potassium Iodide on Thyroid Function and Oxidative Stress in Iodine-Excess Wistar Rats.

The objective of this study was to investigate the effects of organic iodine (3,5-diiodotyrosine, DIT) and inorganic iodine (potassium iodine, KI) on thyroid function and oxidative stress in iodine-excess Wistar rats. Seventy-two Wistar rats were randomly divided into eight groups: normal control (NC), thyroid tablet-induced hyperthyroidism model (HM), low DIT (L-DIT), medium DIT (M-DIT), high DIT (H-DIT), low KI (L-KI), medium KI (M-KI), and high KI (H-KI). All rats were fed ad libitum for 30 days. Morphological changes in the thyroid, absolute and relative weights of the thyroid, thyroid function markers free triiodothyronine (FT3) and free thyroxine (FT4), urinary iodine level, and oxidative stress indicators were measured. Compared to the HM groups, the FT3 and FT4 levels decreased in the L-DIT groups; the thyroid weight and thyroid weight/body weight values decreased markedly in the L-DIT and M-DIT groups; serum superoxide dismutase/malondialdehyde increased markedly; glutathione peroxidase activity increased markedly in the L-DIT groups; and malondialdehyde levels decreased significantly in the M-DIT groups. However, the FT3 and FT4 levels decreased and glutathione peroxidase levels increased significantly in the DIT groups compared to their corresponding KI groups. Additionally, urinary iodine levels increased significantly in both DIT and KI groups, while the highest urinary iodine excretion was showed in the DIT groups among groups. When the addition of iodine with the same doses in iodine-excess rats, although neither DIT nor KI normalized iodine levels in the iodine-excess rats, the DIT did less damage than did KI to thyroid follicular cells. Therefore, DIT rather than KI had a protective effect by balancing the antioxidant system when exposed to supraphysiological iodine. These suggest that DIT may be used as a new alternative iodized salt in the universal salt iodization to avoid the potential damage of surplus KI.

In silico investigation of potential TRAF6 inhibitor from traditional Chinese medicine against cancers.

It has been indicated that tumor necrosis factor receptor-associated factor-6 (TRAF6) will upregulate the expression of hypoxia-inducible factor-1α (HIF-1α) and promote tumor angiogenesis. TRAF6 proteins can be treated as drug target proteins for a differentiation therapy against cancers. As structural disordered disposition in the protein may induce the side-effect and reduce the occupancy for ligand to bind with target protein, PONDR-Fit protocol was performed to predict the disordered disposition in TRAF6 protein before virtual screening. TCM compounds from the TCM Database@Taiwan were employed for virtual screening to identify potent compounds as lead compounds of TRAF6 inhibitor. After virtual screening, the MD simulation was performed to validate the stability of interactions between TRAF6 proteins and each ligand. The top TCM compounds, tryptophan, diiodotyrosine, and saussureamine C, extracted from Saussurea lappa Clarke, Bos taurus domesticus Gmelin, and Lycium chinense Mill., have higher binding affinities with target protein in docking simulation. However, the docking pose of TRAF6 protein with tryptophan is not stable under dynamic condition. For the other two TCM candidates, diiodotyrosine and saussureamine C maintain the similar docking poses under dynamic conditions. Hence, we propose the TCM compounds, diiodotyrosine and saussureamine C, as potential candidates as lead compounds for further study in drug development process with the TRAF6 protein against cancer.

[Thyroid hormones and their precursors I. Biochemical properties]. / Pajzsmirigyhormonok és eloanyagaik I. Biokémiai tulajdonságok.

This paper and the following one (see the next issue of Acta Pharmaceutica Hungarica) survey the biological roles and the related site-specific physico-chemical parameters (basicity and lipophilicity) of the presently known thyroid hormones (thyroxine, liothyronine and reverse liothyronine) and their biological precursors (monoiodotyrosine and diiodotyrosine). Here the literature of the thyroid hormone biochemistry, biosynthesis, plasma- and membrane transport is summarized, focusing on the pH-dependent processes. Biosyntheses of the thyroid hormones take place by oxidative coupling of two iodotyrosine residues catalyzed by thyreoperoxidase in thyreoglobulin. The protonation state of the precursors, especially that of the phenolic OH is crucial for the biosynthesis, since anionic iodotyrosine residues can only be coupled in the thyroid hormone biosyntheses. In the blood more than 99% of the circulating thyroid hormone is bound to plasma proteins among which the thyroxine-binding globulin and transthyretin are crucial. The amphiphilic character of the hormones is assumed to be the reason why their membrane transport is an energy-dependent, transport-mediated process, in which the organic anion transporter family, mainly OATP1C1, and the amino acid transporters, such as MCT8 play important roles. Liothyronine is the biologically active hormone; it binds the thyroid hormone receptor, a type of nuclear receptor. There are two major thyroid hormone receptor (TR) isoforms, alfa (TRalpha) and beta (TRbeta). The activation of the TRalpha is associated with modifications in cardiac behavior, while activation of the TRbeta is associated with increasing metabolic rates, resulting in weight loss and reduction of blood plasma lipid levels. The affinity of the thyroid hormones for different proteins depends on the ionization state of the ligands. The site-specific physico-chemical characterization of the thyroid hormones is of fundamental importance to understand their (patho)physiological behavior and also, to influence their therapeutic properties at the molecular level.

Species-specific lipophilicity of thyroid hormones and their precursors in view of their membrane transport properties.

A total of 30 species-specific partition coefficients of three thyroid hormones (thyroxine, liothyronine, reverse liothyronine) and their two biological precursors (monoiodotyrosine, diiodotyrosine) are presented. The molecules were studied using combined methods of microspeciation and lipophilicity. Microspeciation was carried out by (1)H NMR-pH and UV-pH titration techniques on the title compounds and their auxiliary derivatives of reduced complexity. Partition of some of the individual microspecies was mimicked by model compounds of the closest possible similarity, then correction factors were determined and introduced. Our data show that the iodinated aromatic ring system is the definitive structural element that fundamentally determines the lipophilicity of thyroid hormones, whereas the protonation state of the aliphatic part plays a role of secondary importance. On the other hand, the lipophilicity of the precursors is highly influenced by the protonation state due to the relative lack of overwhelmingly lipophilic moieties. The different logp values of the positional isomers liothyronine and reverse liothyronine represent the importance of steric and electronic factors in lipophilicity. Our investigations provided clear indication that overall partition, the best membrane transport - predicting physico-chemical parameter depends collectively on the site-specific basicity and species-specific partition coefficient. At physiological pH these biomolecules are strongly amphipathic due to the lipophilic aromatic rings and hydrophilic amino acid side chains which can well be the reason why thyroid hormones cannot cross membranes by passive diffusion and they are constituents of biological membranes. The lipophilicity profile of thyroid hormones and their precursors are calculated and depicted in terms of species-specific lipophilicities over the entire pH range.

Differentiation of enantiomeric drugs by iodo-substituted L-amino acid references under electrospray ionization mass spectrometric conditions.

RATIONALE: In chiral differentiation by mass spectrometry, use of a single reference that differentiates various classes of compounds including drugs is ideal, but so far there are no such reports in the literature. We have successfully used iodo-substituted amino acids for the chiral differentiation of ten enantiomeric pairs of drugs. METHODS: To achieve the chiral differentiation, the trimeric Cu complex ion consisting of two chiral reference molecules and an analyte molecule was generated under positive ion electrospray ionization (ESI) conditions and subsequently subjected for collision- induced dissociation (CID) experiments using an LCQ ion trap mass spectrometer. The spectra were recorded under identical experimental conditions for both the enantiomers, and were averages of 30 scans. Cooks' kinetic method and chiral recognition ratio method (CR method) were used to arrive at the R(chiral) /CR values, respectively. RESULTS: The R(chiral) or CR values of the studied drugs are higher for 3,5-diiodo-L-tyrosine as the reference, than for 4-iodo-L-phenylalanine, except for isoproterenol and atenolol. Both the references show the same selectivity (R- or S-selectivity) towards all the studied drugs. With 3,5-diiodo-L-tyrosine as the reference, an R(chiral) value of 12.75 is obtained for DOPA and this is the highest reported value in the literature till now. The suitability of the current method in measuring enantiomeric excess is also demonstrated for DOPA. CONCLUSIONS: The use of 4-iodo-L-phenylalanine or 3,5-diiodo-L-tyrosine as a chiral reference for the chiral differentiation of ten enantiomeric pairs of pharmaceutically important drugs has been demonstrated. The chiral differentiation of pregabalin, tenofovir and pramipexole is reported for the first time. This study shows that it is possible to develop a single chiral reference compound for the differentiation of a group of chiral drugs having some similarities.

Deciphering the peptide iodination code: influence on subsequent gas-phase radical generation with photodissociation ESI-MS.

Iodination of tyrosine was recently discovered as a useful method for generating radical peptides via photodissociation of carbon-iodine bonds by an ultraviolet photon in the gas phase. The subsequent fragmentation behavior of the resulting odd-electron peptides is largely controlled by the radical. Although previous experiments have focused on mono-iodination of tyrosine, peptides and proteins can also be multiply iodinated. Tyrosine and, to a lesser extent, histidine can both be iodinated or doubly iodinated. The behavior of doubly iodinated residues is explored under conditions where the sites of iodination are carefully controlled. It is found that radical peptides generated by the loss of a single iodine from doubly iodinated tyrosine behave effectively identically to singly iodinated peptides. This suggests that the remaining iodine does not interfere with radical directed dissociation pathways. In contrast, the concerted loss of two iodines from doubly iodinated peptides yields substantially different results that suggest that radical recombination can occur. However, sequential activation can be used to generate multiple usable radicals in different steps of an MS(n) experiment. Furthermore, it is demonstrated that in actual peptides, the rate of iodination for tyrosine versus mono-iodotyrosine cannot be predicted easily a priori. In other words, previous assumptions that mono-iodination of tyrosine is the rate-limiting step to the formation of doubly iodinated tyrosine are incorrect.

Preparation and testing of stationary phases and modified capillaries for affinity chromatography and affinity capillary electrophoresis of pepsin.

Three stationary phases have been prepared for affinity liquid chromatography isolation and separation of porcine and human pepsin. The phases contain 3,5-diiodo-L-tyrosine (DIT) bound to the supports HEMA BIO VS, HEMA BIO E and EPOXY TOYOPEARL. These phases have been tested on a model sample of porcine pepsin A and applied to human pepsin. Fractions have been collected and the chymase activity determined in selected analyses. For affinity CE, capillaries have been prepared by modifying the wall with 3-aminopropyltriethoxysilane, followed either by direct binding of DIT, or by binding L-tyrosine that was subsequently iodated. The dissociation constant K(d) has been determined for the pepsin-DIT complex from the changes in the electrophoretic mobilities.

Analysis of thyroglobulin iodination by tandem mass spectrometry using immonium ions of monoiodo- and diiodo-tyrosine.

Peptides containing a monoiodo- or diiodo-tyrosine residue (monoiodo-Y, diiodo-Y) were found to generate abundant immonium ions following collision-induced dissociation at m/z 261.97 and 387.87 Da, respectively. These residue-specific marker ions are between about 140 mDa (monoiodo-Y) and 300 mDa (diiodo-Y) mass deficient relative to any other peptide fragment ions of unmodified peptides, qualifying them as highly specific marker ions for tyrosine iodination when analyzed by high resolution tandem mass spectrometry (MS/MS). Two new iodination sites (Y-364 and Y-2165) were pinpointed in bovine thyroglobulin by MS/MS using these iodotyrosine-specific marker ions and combined tryptic/chymotryptic digestion.

Studies on experimental iodine allergy: 3. Low molecular weight elicitogenic antigens of iodine allergy.

We hypothesize that iodine allergy is an immune response to iodinated self proteins produced in vivo from various iodine-containing chemicals. Since an antigenic determinant of experimental iodine allergy is diiodotyrosine (DIT), we designed low molecular weight DIT derivatives having provocative antigenicity without sensitizing immunogenicity. Tetraiododityrosine and hexaiodotrityrosine provoked dose-dependent skin reactions in guinea pigs previously immunized with iodine. No guinea pigs immunized with hexaiodotrityrosine showed anaphylactic reaction by i.v. challenge with hexaiodotrityrosine and none of their antisera showed positive passive cutaneous anaphylaxis (PCA) reaction in guinea pigs, indicating the non-immunogenic nature of the compound. Erythrosine, one of the color additives having a structure common with DIT, was assessed for its immunological property. Enzyme-linked immunosorbent assay (ELISA) inhibition studies on erythrosine revealed that the inhibitory activity of erythrosine was stronger than that of DIT. Furthermore, erythrosine provoked a PCA reaction in animals sensitized with anti-iodine antisera. In conclusion, hexaiodotrityrosine is thought to be useful for skin testing of iodine allergy without any fear of sensitization to the allergen. Erythrosine was shown to provoke an experimental iodine allergy and, also, the relationships between the new concept of iodine allergy and features of clinical findings of adverse effects by iodocontrast media are discussed.

Design of a pH-sensitive pore-forming peptide with improved performance.

GALA is a 30 residue synthetic peptide designed to interact with membranes in a pH-sensitive manner, with potential applications for intracellular drug and gene delivery. Upon reduction of the pH from neutral to acidic, GALA switches from random coil to alpha-helix, inserts into lipid bilayers, and forms oligomeric pores of defined size. Its simple sequence and well-characterized behavior make the peptide an excellent starting point to explore the effects of sequence on structure, pH sensitivity, and membrane affinity. We describe synthesis and characterization of two derivatives of GALA, termed GALAdel3E and YALA. GALAdel3E has a deletion of three centrally located glutamate residues from GALA, while YALA replaces one glutamate residue with the unusual amino acid 3,5-diiodotyrosine. Both derived peptides retain pH sensitivity, showing no ability to cause leakage of an encapsulated dye from unilamellar vesicles at pH 7.4 but substantial activity at pH 5. Unlike GALA, neither peptide undergoes a conformational change upon reduction of the pH, remaining alpha-helical throughout. Interestingly, the pH at which the peptides activate is shifted, with GALA becoming active at pH approximately 5.7, GALAdel3E at pH approximately 6.2, and YALA at pH approximately 6.7. Furthermore, the peptides GALAdel3E and YALA show improved activity compared with GALA for cholesterol-containing membranes, with YALA retaining the greatest activity. Improved activity in the presence of cholesterol and onset of activity in the critical range between pH 6 and 7 may make these peptides useful in applications requiring intracellular delivery of macromolecules, such as gene delivery or anti-cancer treatments.

Affinity chromatography of porcine pepsin on different types of immobilized 3,5-diiodo-L-tyrosine.

The preparation of affinity sorbents containing immobilized iodinated derivatives of L-tyrosine for the affinity chromatography of porcine pepsin is described. The ligand was coupled either to Sepharose 4B or bead cellulose after the divinylsulfone activation or to Sepharose 4B after the activation with 2,4,6-trichloro-1,3,5-triazine. The highest capacity for porcine pepsin was found in the case of 3,5-diiodo-L-tyrosine coupled to divinylsulfone-activated Sepharose.

Conformational dynamics of thyroid hormones by variable temperature nuclear magnetic resonance: the role of side chain rotations and cisoid/transoid interconversions.

1H NMR spectra of the thyroid hormone thyroxine recorded at low temperature and high field show splitting into two peaks of the resonance due to the H2,6 protons of the inner (tyrosyl) ring. A single resonance is observed in 600 MHz spectra at temperatures above 185 K. An analysis of the line shape as a function of temperature shows that the coalescence phenomenon is due to an exchange process with a barrier of 37 kJ mol-1. This is identical to the barrier for coalescence of the H2',6' protons of the outer (phenolic) ring reported previously for the thyroid hormones and their analogues. It is proposed that the separate peaks at low temperature are due to resonances for H2,6 in cisoid and transoid conformers which are populated in approximately equal populations. These two peaks are averaged resonances for the individual H2 and H6 protons. Conversion of cisoid to transoid forms can occur via rotation of either the alanyl side chain or the outer ring, from one face of the inner ring to the other. It is proposed that the latter process is the one responsible for the observed coalescence phenomenon. The barrier to rotation of the alanyl side chain is > or = 37 kJ mol-1, which is significantly larger than has previously been reported for Csp2-Csp3 bonds in other Ph-CH2-X systems. The recent crystal structure of a hormone agonist bound to the ligand-binding domain of the rat thyroid hormone receptor (Wagner et al. Nature 1995, 378, 690-697) shows the transoid form to be the bound conformation. The significant energy barrier to cisoid/transoid interconversion determined in the current study combined with the tight fit of the hormone to its receptor suggests that interconversion between the forms cannot occur at the receptor site but that selection for the preferred bound form occurs from the 50% population of the transoid form in solution.

Preparation of radiolabeled peptides via an iodine exchange reaction.

Peptides labeled with radioactive 125I can be detected at the extremely low concentrations necessary for receptor binding studies and medical applications. Traditional methods of iodination often lead to inactive peptides due to excessive iodination, nonspecific iodination, or oxidative damage to the peptide. In this work these disadvantages are circumvented by labeling 127I containing peptides of a predetermined biological activity using a radioactive 125I exchange reaction with Na125I. Specific radioactivity up to 9.8 Ci/mmol was reached in a simple and efficient procedure.

Development of a new method for the determination of thyroxine in serum based on isotope dilution gas chromatography mass spectrometry.

A new gas chromatographic/mass spectrometric method in combination with isotope dilution for the determination of thyroxine in serum is described. Special attention was paid to the methylation step of thyroxine, which was investigated using methanolic HCl, dimethylformamide/dimethylacetal and diazomethane, the latter giving the best results in terms of reproducible isotope ratios. For internal standardization, (13C6)-thyroxine was dissolved in fraction V human albumin solution (70 g l-1). The internal standard-in-albumin solution was mixed with known amounts of thyroxine standard, dissolved in 0.05 M Na2HPO4 buffer at pH 11.6, to give isotope ratios of 0.75, 1.00 and 1.25. The same internal standard solution was also used for isotope dilution of the unknown serum samples. The volume of serum was adapted to give a 1:1 isotope ratio. Sample pretreatment consisted of protein precipitation and a two-step liquid/liquid extraction procedure. After methylation of unlabelled and labelled thyroxine with diazomethane and perfluoroacylation with pentafluoropropionic anhydride and heptafluorobutyric anhydride, respectively, mass spectrometric monitoring was done at m/z 951/957 and 1001/1007. Quantitative determination of thyroxine in five serum samples in duplicate, during three consecutive days, showed a mean overall imprecision of 1.0% and a deviation of +0.4% from the target value as determined by a definitive method.

A comparison of thyrotropin- and dibutyryl cyclic AMP- induced thyroglobulin iodination in cultured human thyroid cells.

The relative degree of 125-I labelling of thyroglobulin-- bound mono-iodotyrosine (MIT) and di-iodotyrosine (DIT) in isolated, cultured human thyroid cells has been compared following exposure of 125-I supplemented cells to 100 mU/ml of bovine thyrotropin (TSH) or 1.0 mM dibutyryl cyclic AMP (dBcAMP) for 96 hours. Pronase digestion of the lysed cells and Sephadex G-10 fractionation of the digested lysates revealed a predominance of [125-I]MIT over [125-I]DIT in both sets of experimental cells as well as in controls. Levels of [125-I]DIT, however, were only enhanced above control values in cells incubated with TSH. These findings suggest that an increase in availability of intracellular iodide, following cellular exposure to TSH, may facilitate a preferential synthesis of DIT relative to that of MIT. This theory offers an explanation for the differential effects of TSH and dibutyryl cyclic AMP on the levels of newly--synthesised T4 recovered from the cells used in this study, and from the culture medium in a previous investigation.