RESUMO
Many halogenated organic contaminants (HOCs) are considered endocrine disruptors and affect the hypothalamic-pituitary-thyroid axis, often by interfering with circulating levels of thyroid hormones (THs). We investigated one potential mechanism for TH disruption, inhibition of sulfotransferase activity. One of the primary roles of TH sulfation is to support the regulation of biologically active T3 through the formation of inactive THs. We investigated TH sulfotransferase inhibition by 14 hydroxylated polybrominated diphenyl ethers (OH BDEs), BDE 47, triclosan, and fluorinated, chlorinated, brominated, and iodinated analogues of 2,4,6-trihalogenated phenol and bisphenol A (BPA). A new mass spectrometry-based method was also developed to measure the formation rates of 3,3'-T2 sulfate (3,3'-T2S). Using pooled human liver cytosol, we investigated the influence of these HOCs on the sulfation of 3,3'-T2, a major substrate for TH sulfation. For the formation of 3,3'-T2S, the Michaelis constant (Km) was 1070 ± 120 nM and the Vmax was 153 ± 6.6 pmol min(-1) (mg of protein)(-1). All chemicals investigated inhibited sulfotransferase activity with the exception of BDE 47. The 2,4,6-trihalogenated phenols were the most potent inhibitors followed by the OH BDEs and then halogenated BPAs. The IC50 values for the OH BDEs were primarily in the low nanomolar range, which may be environmentally relevant. In silico molecular modeling techniques were also used to simulate the binding of OH BDE to SULT1A1. This study suggests that some HOCs, including antimicrobial chemicals and metabolites of flame retardants, may interfere with TH regulation through inhibition of sulfotransferase activity.
Assuntos
Retardadores de Chama/farmacologia , Fenóis/farmacologia , Sulfotransferases/metabolismo , Glândula Tireoide/enzimologia , Compostos Benzidrílicos/análise , Compostos Benzidrílicos/metabolismo , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Di-Iodotironinas/análise , Di-Iodotironinas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Retardadores de Chama/análise , Retardadores de Chama/metabolismo , Éteres Difenil Halogenados/análise , Éteres Difenil Halogenados/química , Éteres Difenil Halogenados/metabolismo , Éteres Difenil Halogenados/farmacologia , Humanos , Cinética , Fígado/metabolismo , Simulação de Acoplamento Molecular , Fenóis/análise , Fenóis/química , Fenóis/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Sulfatos/química , Sulfatos/metabolismo , Sulfotransferases/antagonistas & inibidores , Espectrometria de Massas em TandemRESUMO
PURPOSE: The direct aqueous photolysis of the thyroid hormone levothyroxine (T(4)) has been studied. METHODS AND RESULT: One of the major photoproducts, i.e., 4-[4-(2-amino-2-carboxy-ethyl)-2,6-diiodo-phenoxy]-penta-2,4-dienoic acid (P1), was isolated by liquid chromatography and structurally assigned by mass spectrometric (MS) and nuclear magnetic resonance spectroscopic methods. The identity of a second major product, i.e., 3,5-diiodo-L: -thyrosine (P3), was confirmed through access to a commercially available standard. Furthermore, the structures of three additional transformation products are proposed on the basis of data obtained by high-resolution MS analyses. UV absorption spectra were determined for T(4) and the two photoproducts P1 and P3. Disappearance quantum yields were calculated for T(4) (Ï = 0.014 at pH 12) and P3 (Ï = 0.024 at pH 12 and Ï = 0.010 at pH 8.5), whereas the compound P1 was found to be stable under the studied conditions (T(1/2) = 600 min). CONCLUSION: The results indicate that solar UV light may have a significant impact on the fate of T(4) in the aquatic environment.
Assuntos
Fotólise , Tiroxina/análise , Tiroxina/química , Cromatografia Líquida de Alta Pressão , Di-Iodotironinas/análise , Di-Iodotironinas/química , Monitoramento Ambiental , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Raios Ultravioleta , Água/química , Poluentes da Água/análise , Poluentes da Água/metabolismo , Poluição da Água/análiseRESUMO
Diiodothyronines 3,5-diiodothyronine (3,5-T2), 3',5'-diiodothyronine (3',5'-T2), and 3,3'-diiodothyronine (3,3'-T2) are important metabolites of 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3; reverse T3). In this paper, a novel and rapid method for identifying and quantifying 3,5-T2, 3',5'-T2 and 3,3'-T2 has been introduced using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Fragmentation patterns were proposed on the basis of our data obtained by ESI-MS/MS. MS2 spectra in either negative ionization mode or positive ionization mode can be used to differentiate 3,5-T2, 3',5'-T2 and 3,3'-T2. On the basis of the relative abundance of fragment ions in MS2 spectra under the positive ionization mode, quantification of the 3,5-T2, 3',5'-T2 and 3,3'-T2 isomers in mixtures is also achieved without prior separation.
Assuntos
Di-Iodotironinas/análise , Di-Iodotironinas/química , Di-Iodotironinas/sangue , Humanos , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por ElectrosprayAssuntos
Di-Iodotironinas/metabolismo , Doenças da Glândula Tireoide/metabolismo , Adulto , Di-Iodotironinas/análise , Feminino , Sangue Fetal , Humanos , Recém-Nascido , Gravidez/sangue , Radioimunoensaio , Valores de Referência , Doenças da Glândula Tireoide/diagnóstico , Testes de Função TireóideaRESUMO
OBJECTIVE: The diagnosis of fetal hypothyroidism is based at present on measurements of TSH and free thyroxine (FT4) in fetal blood samples obtained by cordocentesis. The measurement of maternal serum and urinary concentrations of compound W, immunologically similar to but chromatographically distinct from diiodothyronine sulfate (T2S), has been advocated as a new possible marker for fetal hypothyroidism. DESIGN: In this paper, we measured serum compound W levels in 84 pregnant women, 20 with and 64 without thyroid disorders before and during specific treatment. Compound W was also assessed in fetal blood obtained by cordocentesis from 49 normal fetuses and 4 fetuses with suspected hypothyroidism due to transplacental passage of propylthiouracil (PTU). Compound W levels were measured by T2S RIA in maternal and fetal serum. To assess the possible usefulness of 3, 5,3'-triiodothyroacetic acid (TRIAC) for therapy of fetal hypothyroidism we evaluated the transplacental passage of TRIAC by administering the drug to four pregnant women before therapeutic abortion. RESULTS: In normal pregnancies, both maternal and fetal compound W levels increased progressively during gestation with a significant direct correlation (P<0.001, in both mothers and fetuses). Moreover, a significant positive correlation was observed between fetal compound W and fetal FT4 values (P<0.005), whereas no correlation was observed between maternal serum compound W and maternal FT4 in either euthyroid or hyperthyroid women, suggesting the fetal origin of compound W. The hypothyroid fetuses of PTU-treated mothers showed low compound W levels, and maternal compound W values were in the low normal range and did not show the typical increase during progression of gestation. A significant increase of maternal compound W was observed when the PTU dose was reduced. TRIAC was documented to cross the placental barrier and the treatment of a hyperthyroid pregnant woman on PTU caused the high fetal TSH levels and goiter to normalize. CONCLUSIONS: Serial measurements of 3,3'-T2S crossreactive materials (compound W and 3, 3'-diiodothyroacetic acid sulfate) in maternal blood and the administration of TRIAC to the mother may represent a useful and safe alternative to invasive techniques for the diagnosis and therapy of fetal hypothyroidism.
Assuntos
Biomarcadores/análise , Di-Iodotironinas/análise , Doenças Fetais/diagnóstico , Hipotireoidismo/diagnóstico , Cordocentese , Di-Iodotironinas/sangue , Di-Iodotironinas/urina , Feminino , Sangue Fetal/química , Doenças Fetais/induzido quimicamente , Doenças Fetais/tratamento farmacológico , Idade Gestacional , Humanos , Hipotireoidismo/induzido quimicamente , Hipotireoidismo/tratamento farmacológico , Troca Materno-Fetal , Placenta/metabolismo , Gravidez , Propiltiouracila/efeitos adversos , Tireotropina/sangue , Tiroxina/sangue , Tri-Iodotironina/administração & dosagem , Tri-Iodotironina/análogos & derivados , Tri-Iodotironina/farmacocinética , Tri-Iodotironina/uso terapêuticoRESUMO
In this article we describe the development of a highly sensitive, accurate, and reproducible RIA for the measurement of 3,3'-diiodothyronine (3,3'-T2) in human serum and brain tissue. The detection limits were 1.8 fmol/g and 1.5 pmol/L in human brain tissue and serum, respectively. Serum concentrations of 3,3'-T2 were measured in 4 groups of patients with nonthyroidal illnesses (NTI), i.e. brain injuries (n = 15), sepsis (n = 24), liver disease (n = 22), and brain tumors (n = 23). The mean serum concentration of 3,3'-T2 in 62 healthy controls was 46.6 +/- 20.0 pmol/L. 3,3'-T2 levels declined significantly with increasing age. They were significantly lower in patients with brain injury (34.2 +/- 19.4 pmol/L; P = 0.006), were at the upper limit of normal in patients with sepsis (57.0 +/- 36.9 pmol/L; P = 0.06), and were elevated in patients with liver disease (72.6 +/- 56.7 pmol/L; P = 0.04) and brain tumors (89.0 +/- 40.9 pmol/L; P = 0.01). The serum levels of T3 were significantly lower than those in controls in all 4 patient groups. Serum concentrations of 3,3'-T2 were significantly enhanced in 9 patients with hyperthyroidism (85.4 +/- 43.0 pmol/L; P = 0.01) and were reduced in 12 patients with hypothyroidism (14.9 +/- 9.2 pmol/L; P = 0.001). In both normal brain tissue, obtained either intraoperatively or excised postmortem, and brain tumors, the concentrations of 3,3'-T2 ranged between 50-300 fmol/g. In healthy controls, 2 different forms of acute stress (sleep deprivation and delivering a lecture) significantly increased serum levels of T4 and T3, but did not affect those of 3,3'-T2 or 3,5-T2. In conclusion, our results show that, contrary to expectation, a low T3 syndrome in NTI is not always associated with low serum concentrations of 3,3'-T2. The production of 3,3'-T2 in NTI seems to be regulated in a disease-specific manner, resulting in unchanged, reduced, or elevated hormone concentrations.
Assuntos
Neoplasias Encefálicas/sangue , Di-Iodotironinas/sangue , Estresse Fisiológico/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Química Encefálica , Lesões Encefálicas/sangue , Di-Iodotironinas/análise , Feminino , Humanos , Hepatopatias/sangue , Masculino , Pessoa de Meia-Idade , Valores de Referência , Sepse/sangue , Doenças da Glândula Tireoide/sangue , Hormônios Tireóideos/sangueRESUMO
This study reports the development of a highly sensitive and reproducible RIA for the measurement of 3,5-diiodothyronine (3,5-T2) in human serum and tissue. The RIA employs 3-bromo-5-[125I]iodo-L-thyronine (3-Br-5-[125I]T1) as tracer, which was synthesized carrier free by an interhalogen exchange from 3,5-dibromo-L-thyronine (3,5-Br2T0). The detection limits were 1.0 fmol/g and 0.8 pmol/L in human brain tissue and serum, respectively. T3, diiodothyroacetic acid, and 3-monoiodothyronine cross-reacted with a 3,5-T2 antibody to the extent of 0.06%, 0.13%, and 0.65%, respectively. Serum concentrations of 3,5-T2 were measured in 62 healthy controls and 4 groups of patients with nonthyroidal illness, i.e. patients with sepsis (n = 24), liver diseases (n = 23), head and/or brain injury n = 15), and brain tumors (n = 21). The mean serum level of 3,5-T2 in the healthy subjects was 16.2 +/- 6.4 pmol/L. Concentrations of 3,5-T2 were significantly elevated in patients with sepsis (46.7 +/- 48.8 pmol/L; P < 0.01), liver diseases (24.8 +/- 14.9 pmol/L; P < 0.01), head and/or brain injury (24.1 +/- 11.3 pmol/L; P < 0.05), and brain tumors (21.6 +/- 4.8 pmol/L; P < 0.01). In all 4 patient groups, serum levels of T3 were significantly reduced, confirming the existence of a low T3 syndrome in these diseases. Serum concentrations of 3,5-T2 were significantly elevated in patients with hyperthyroidism (n = 9) and were reduced in patients with hypothyroidism (n = 8). The levels of T4, T3, and 3,5-T2 were measured in normal human tissue samples from the pituitary gland and various brain regions and in brain tumors. In normal brain tissue, the concentrations of 3,5-T2 ranged between 70-150 fmol/g, and the ratio of T3 to 3,5-T2 was approximately 20:1. In brain tumors, however, T3 levels were markedly lower, resulting in a ratio of T3 to 3,5-T2 of approximately 1:1. Recent findings suggest a physiological, thyromimetic role of 3,5-T2, possibly stimulating mitochondrial respiratory chain activity. Should this prove to be correct, then the increased availability of 3,5-T2 in nonthyroidal illness may be one factor involved in maintaining clinical euthyroidism in patients with reduced serum levels of T3 during nonthyroidal illness.
Assuntos
Neoplasias Encefálicas/sangue , Di-Iodotironinas/sangue , Radioimunoensaio/métodos , Astrocitoma/metabolismo , Lesões Encefálicas/sangue , Neoplasias Encefálicas/metabolismo , Traumatismos Craniocerebrais/sangue , Di-Iodotironinas/análise , Glioblastoma/metabolismo , Humanos , Hepatopatias/sangue , Radioimunoensaio/estatística & dados numéricos , Valores de Referência , Sensibilidade e Especificidade , Sepse/sangue , Tiroxina/sangue , Tri-Iodotironina/análise , Tri-Iodotironina/sangueRESUMO
We describe a new method for the separation, identification and quantitation of iodotyrosines and iodothyronines [3-monoiodo-L-tyrosine (MIT), 3,5-diiodo-L-tyrosine (DIT), L-thyronine (T0), 3,5-diiodo-L-thyronine (T2), 3,5,3'-triiodo-L-thyronine (T3), reverse 3,3',5'-triiodo-L-thyronine (rT3) and 3,3',5,5'-tetraiodo-L-thyronine (T4)]. Reversed-phase high-performance liquid chromatography (RP-HPLC) was performed on a Nucleosil C8 column with photodiode-array UV-Vis detection. A clearly defined elution profile was obtained of each iodoamino acid (iodotyrosines and iodothyronines) using a linear gradient from 20 to 80% phase B (90% acetonitrile, 10% water, 0.1% TFA), phase A (water, 0.1% TFA, pH 2.0) eluted over 40 min. Iodoamino acid composition was determined, taking into account retention times and spectral characteristics. Thyroid protein samples were digested enzymatically and the complex mixture of IAA was then injected onto the RP-HPLC system. A photodiode-array detector with a dynamic range in the UV-Vis region was used in the HPLC system to monitor the absorbance at different wavelengths continuously, collecting data which were compared with standard samples. Each IAA was quantitated using linear calibration curves obtained at 280 nm. This method allowed identification and quantitation of iodoamino acids from diverse sources in the range 2-500 ng, avoiding the need to radiolabel samples. The technique was tested with in vitro iodinated and non-iodinated human thyroglobulin and the recoveries ranged from 84 to 91%.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Monoiodotirosina/análise , Tireoglobulina/química , Tironinas/análise , Tiroxina/análise , Animais , Calibragem , Di-Iodotironinas/análise , Radioisótopos do Iodo , Modelos Lineares , Ratos , Espectrofotometria Ultravioleta , Tireoglobulina/análise , Tireoglobulina/biossíntese , Glândula Tireoide/química , Tri-Iodotironina/análiseRESUMO
Previous work from this laboratory has shown that the thyroid gland of the fetal pig begins to function at about day 46-47 (0.40-0.415 fraction of gestational age). Sera from fetuses contain lower thyroxine (T4), 3,3',5-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3) concentrations than maternal sera, except for about 2 weeks before term. The fetal T4 metabolism is dominated by the 5'-monodeiodinating activity (5'-MD). In the present study we measured the iodothyronines content, and the outer (5'-MD) and inner (5-MD) monodeiodinases activity, in homogenates of the placenta. The pig placenta, which is of the epitheliochorial type, was separated into the fetal and the maternal part. The concentrations of T4, T3 and rT3 were lower, and the deiodinating activity of 5'-MD and 5-MD higher, in the fetal than in the maternal placenta. The fetal placenta not only deiodinated more actively T4 to T3 and T4 to rT3, but degraded T3 to 3,3'-diiodothyronine (3,3'-T2) more actively than rT3 to 3,3'-T2. Such divergent deiodinating activity of T4 to T3, T3 to 3,3'-T2 and rT3 to 3,3'-T2 might favor establishing a relatively high and constant rT3 concentrations in fetal and maternal placentas, and a lower T3 in the fetal placenta. The inner ring deiodinating activity (excluding a day before parturition) was always more active in the fetal placenta, while the outer ring deiodinations varied in this respect, depending on the gestation stage. These results support the hypothesis that in the fetal pig, enzymatic deiodination of thyroid hormones forms a barrier which reduces transplacental passage of the hormones and that the fetal part of the placenta is the primary factor in the mechanism regulating the hormonal transfer. In spite of the presence of the barrier, there is an adequate maternal supply of thyroid hormones to the fetus in early gestation, which suggests that the enzymatic mechanism is influenced in some way by the thyroid status of the fetus.
Assuntos
Iodeto Peroxidase/metabolismo , Placenta/metabolismo , Prenhez/metabolismo , Suínos/metabolismo , Hormônios Tireóideos/metabolismo , Animais , Di-Iodotironinas/análise , Di-Iodotironinas/metabolismo , Feminino , Feto/metabolismo , Iodeto Peroxidase/análise , Placenta/enzimologia , Gravidez , Radioimunoensaio , Hormônios Tireóideos/análise , Tiroxina/análise , Tiroxina/metabolismo , Tri-Iodotironina/análise , Tri-Iodotironina/metabolismo , Tri-Iodotironina Reversa/análise , Tri-Iodotironina Reversa/metabolismoRESUMO
Thyroxine (T4), 3,3',5'-tri-iodothyronine (reverse T3; rT3) and di-iodothyronines (3,3'-T2 and 3',5'-T2) were measured in pig amniotic fluid (AF) and allantoic fluid (Al) between 32 and 113 days of normal pregnancy. Low but measurable quantities of T4 in AF and Al (2.1 +/- 0.3 and 3.2 +/- 0.5 nmol/l respectively) were found before the onset of fetal thyroid gland function, which indicates the maternal source of T4. The presence of rT3 (55.8 +/- 4.1 pmol/l in AF and 49.8 +/- 5.3 pmol/l in Al), 3,3'-T2 (45.5 +/- 0.6 pmol/l in AF and 49.2 +/- 9.2 pmol/l in Al) and 3',5'-T2 (20.8 +/- 2.6 pmol/l in AF and 24.0 +/- 2.2 pmol/l in Al) may be attributed to the monodeiodinase system already active in fetal pig tissues in early pregnancy, as demonstrated previously. T3 concentration was undetectable in both AF and Al. An approximately twofold increase in the levels of T4, rT3 and T2s in AF and Al at mid-gestation was observed. T4 and rT3 in AF showed a positive correlation with protein concentrations. AF rT3 concentration (but not T4) correlated with rT3 in the cord and maternal serum. The 3,3'-T2 and 3',5'-T2 in AF and Al showed parallel changes to rT3, while the rT3/3,3'-T2 and rT3/3',5'-T2 molar ratios remained constant. T4 concentrations in AF and Al were markedly lower than in corresponding maternal and fetal serum; the rT3 concentration in Al was equal to that in AF and two to four times lower than in fetal serum. In spite of differences between serum hormone patterns in the pig and human near term, iodothyronine concentrations in AF showed some similarities, mainly the following: undetectable T3, a strong correlation between rT3, T4 and AF total protein and the presence of 3,3'-T2 and 3',5'-T2 in measurable levels. Comparative data for Al, except the ones in the present study in the pig, are not available.
Assuntos
Alantoide/metabolismo , Líquido Amniótico/metabolismo , Prenhez/metabolismo , Suínos/metabolismo , Hormônios Tireóideos/metabolismo , Alantoide/química , Líquido Amniótico/química , Animais , Di-Iodotironinas/análise , Di-Iodotironinas/metabolismo , Feminino , Idade Gestacional , Isomerismo , Gravidez , Radioimunoensaio , Hormônios Tireóideos/análise , Tiroxina/análise , Tiroxina/metabolismo , Tri-Iodotironina/análise , Tri-Iodotironina/metabolismo , Tri-Iodotironina Reversa/análise , Tri-Iodotironina Reversa/metabolismoAssuntos
Tironinas/análise , Tirosina/análise , Cromatografia Líquida de Alta Pressão , Di-Iodotironinas/análise , Di-Iodotironinas/urina , Di-Iodotirosina/análise , Di-Iodotirosina/urina , Espectrofotometria Ultravioleta , Tireoglobulina/análise , Tironinas/urina , Tri-Iodotironina/análise , Tri-Iodotironina/urina , Tirosina/urinaRESUMO
A radioimmunoassay for 3,5-diiodothyronine (3,5-T2) was developed using inner ring labeled 125l-3,5-T2 and a 3,5-T2 antibody produced in rabbits by immunization with 3,5-T2 coupled to human albumin. Separation of free and bound 3,5-T2 was achieved by ascending ion-exchange wick-chromatography. The detection limit was 4 pmol/l. The only important cross-reaction was with T3. It amounted to 0.11 % (mol/mol) and contributed only little to the 3,5-T2 measured in thyroid hydrolysate and thyroid effluent. It was ascertained that no significant loss or generation of 3,5-T2 took place during storage of thyroid effluent samples and during hydrolysis of thyroid homogenate. In four experiments employing eight thyroid lobes, 3,5-T2 was measured in hydrolysate and effluent from perfused dog thyroid lobes during single passage perfusion with a synthetic hormone free medium. During perfusion with control medium 3,5-T2 in effluent was stable around 40 pmol/l. Infusion of 100 microunits/ml TSH induced after a latency period of approximately 20 min a gradual increase in 3,5-T2 release to a level of approximately 180 pmol/l. 3,5-T2 in thyroid hydrolysate was 1.47 +/- 0.05 pmol/mg thyroid wet weight (mean +/- SD). There did not seem to be any major generation of 3,5-T2 by intrathyroidal iodothyronine deiodination. Thus thyroidal secretion of 3,5-T2 is very low and would probably not contribute significantly to the amounts of circulating 3,5-T2.
Assuntos
Di-Iodotironinas/análise , Glândula Tireoide/análise , Tironinas/análise , Animais , Cromatografia por Troca Iônica , Di-Iodotironinas/metabolismo , Cães , Técnicas In Vitro , Perfusão , Propiltiouracila/farmacologia , Radioimunoensaio , Glândula Tireoide/efeitos dos fármacos , Tironinas/metabolismoRESUMO
Changes are described in the content of butanol-extracted iodine in the blood serum and total iodine in thyroid tissue of white rat males under ultrasonic effect of various intensity (0.2, 0.6, 1.0 Watt/cm2) and analysed at different time intervals after ultrasound application. A decrease in total iodine content in thyroid glands (at initial level 0.315 mg%) and an increase of butanol-extracted iodine content in the blood serum were observed in all the experimental groups. The data obtained compared with the earlier found increase of the thyroid hormone content in the serum and thyreotropic hormone at a decreased content of thyroxins in the thyroid gland seem to point to the activation of the thyroid function after the application of ultrasound.
Assuntos
Glândula Tireoide/fisiologia , Ultrassom , Animais , Di-Iodotironinas/análise , Iodo/análise , Masculino , Ratos , Testes de Função Tireóidea , Glândula Tireoide/análise , Tri-Iodotironina/análiseAssuntos
Di-Iodotironinas/análise , Iodeto Peroxidase/metabolismo , Monoiodotirosina/análise , Peroxidases/metabolismo , Glândula Tireoide/enzimologia , Tironinas/análise , Tri-Iodotironina/análise , Animais , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia em Papel/métodos , Cinética , Ovinos , Espectrofotometria/métodos , Tiroxina/análiseRESUMO
We describe a 10-min reversed-phase "high-pressure' liquid-chromatographic procedure for measuring tyrosine, monoiodotyrosine, diiodotyrosine, 3,5-diiodothyronine, 3,5,3'-triiodothyronine, 3,3',5'-triiodothyronine, and thyroxine. Resolution and quantitation of a mixture of these amino acids were excellent on LiChrosorb (Altex) RP-8 with isocratic elution (1.5 mL/min) with acetonitrile/water/glacial acetic acid (50/49/1 by vol). As little as 100 ng of each iodoamino acid could be detected and quantitated with a conventional 1-cm, flow-through spectrophotometric (254-nm) detector coupled to a 10-mV strip-chart recorder. Analyses for monoiodotyrosine, diiodotyrosine, 3,5,3'-triiodothyronine, and thyroxine in hog and beef thyroglobulin hydrolysates (sequential digestion with pronase and aminopeptidase) agreed well with results by anion-exchange chromatography and by competitive radioassays. To prevent interference by tryptophan in the analysis for diiodotyrosine, we batch-separated the iodoamino acids by anion-exchange chromatography before the procedure. The procedure we describe seems generally useful for detection and quantitation of thyroid hormones, thyroid hormone metabolites, and iodotyrosines.
Assuntos
Monoiodotirosina/análise , Tireoglobulina/análise , Tironinas/análise , Tiroxina/análise , Animais , Bovinos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , Di-Iodotironinas/análise , Ensaio Radioligante , Especificidade da Espécie , Suínos , Tri-Iodotironina/análiseAssuntos
Di-Iodotironinas/análise , Radioimunoensaio , Glândula Tireoide/análise , Tironinas/análise , Líquido Amniótico/análise , Di-Iodotironinas/sangue , Feminino , Sangue Fetal/análise , Doença de Graves/metabolismo , Humanos , Hipertireoidismo/sangue , Hipotireoidismo/sangue , Gravidez , Tireoglobulina/análise , Tiroxina/análise , Tri-Iodotironina/análiseRESUMO
Some iodoamino acids have been separated and determined by high pressure liquid chromatography on octadecylsilane reverse phase packing with a mobile phase consisting of methanol, potassium phosphate monobasic, and orthophosphoric acid at 44 degrees C. The method separates and quantitates mixtures of 3,5-diiodothyronine, liothyronine, isoliothyronine, and levothyroxine. The procedure provides an accurate, sensitive, and rapid estimation of decomposition and/or impurities in standards in approximately 30 min, at the less than 75 pmole level, using an ultraviolet detector at 254 nm.
Assuntos
Tiroxina/análise , Tri-Iodotironina/análise , Aminoácidos/análise , Cromatografia Líquida de Alta Pressão , Di-Iodotironinas/análise , Contaminação de MedicamentosRESUMO
Amniotic fluid concentrations of 3,3',5'-tri-iodothyronine (rT3), 3,3'-Di-iodothyronine (3,3'-T2), 3,5,3'-tri-iodothyronine (T3) and T4 were studied in 384 women during normal and complicated pregnancy. An inverse correlation was observed between decreasing rT3 and increasing 3,3'-T2 concentrations in amniotic fluid with gestational age. The mean rT3 level in normal pregnancy was 2.81 nmol/1 at 12-20 weeks and decreased significantly to 1.06 nmol/1 at 36-42 weeks of gestation. The mean 3,3'-T2 concentration was 49.1 pmol/1 at12-20 weeks increasing to 119 pmol/1 at 36-42 weeks. The mean T4 value of 3.83 nmol/1 at 12-20 weeks was about half that of later periods. The T3 concentration in a random sample of 45 amniotic fluids ranged from less than 28 to 370 pmol/1 (mean 102 pmol/1). The mean rT3, 3,3'-T2 and T4 values measured in patients with intra-uterine malnutrition, gestation diabetes, tocolysis, placental insufficiency and rhesus incompatibility at 31-40 weeks of gestation were not significantly different from those in uncomplicated pregnancy. Significantly decreased rT3 and T4 concentrations were found in toxaemia. From the results obtained in complicated pregnancy it may be concluded that measurements of iodothyronines, especially rT3, in amniotic fluid have insignificant diagnostic value in the recognition of intra-uterine lesions with the probable exception of fetal hypothyroidism. The analysis of the dependence of iodothyronine concentrations on the gestational age showed a maximum of rT3 and T4 levels between 20 and 30 weeks of pregnancy. This marked rise of iodothyronine concentrations in amniotic fluid at mid-gestation may be due to the onsetting maturation of the hypothalamic-pituitary-thyroid control system of the fetus.
Assuntos
Líquido Amniótico/análise , Complicações na Gravidez/metabolismo , Gravidez , Tiroxina/análise , Tri-Iodotironina Reversa/análise , Tri-Iodotironina/análise , Adulto , Di-Iodotironinas/análise , Feminino , Idade Gestacional , HumanosRESUMO
The present report describes a RIA for 3',5'-diiodothyronine (T2) that can be performed on unextracted serum and which has a lower limit of detectability of 2 ng/dl. Cross-reactivity with other iodothyronines was negligible, except for rT3 which began to demonstrate cross-reactivity when rT3 levels were elevated to 180 ng/dl. Employing this RIA for T2, we have determined that 83 healthy individuals had a mean (+/-SE) serum T2 concentration of 5.0 +/- 0.3 ng/dl, thyrotoxic subjects (n = 12) had a mean T2 level that was elevated to 10.8 +/- 0.8 ng/dl, and each of 6 hypothyroid subjects had undetectable (less than 2 ng/dl) concentrations. Athyreotic patients (n = 8), receiving 0.4 mg T4 daily, had serum T2 concentrations of 15.0 +/- 3.0 ng/dl. Fasting in obese subjects was associated with an increase in serum T2 to 6.9 +/- 0.6 ng/dl from a basal level of 4.4 +/- 0.4 ng/dl in the fed state (P less than 0.01). Despite the fact that rT3 levels may be elevated in amniotic fluid and that rT3 is expected to represent the major source from which extrathyroidal T2 arises, T2 levels were low in amniotic fluid, being undetectable (less than 2 ng/dl) in 9 of 19 samples; the mean (+/-SE) T2 concentration in the 10 detectable samples was 5.4 +/- 1 ng/dl. These data indicate T2 is a normal component of serum and that the majority of serum T2 is probably derived from peripheral conversion. Furthermore, these observations suggest that situations associated with elevated rT3 levels (e.g. thyrotoxicosis and fasting) may also have increased T2 values.