Direct immunoassay of triiodothyronine in human serum

Larsen, P. Reed

doi:10.1172/jci107000

Cited by 209 publications

(72 citation statements)

References 19 publications

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“…Four hundredmilliliter samples of amniotic or allantoic fluid were used with 300 p g ANS/tube as described for human amniotic fluid (20). For the standard curves of the allantoic and amniotic fluid assays, T, and T, free fluid was prepared as described by Larsen (14) for human serum. Recovery of T, added to T, free amniotic fluid ranged from 96 to 102%.…”

Section: Methodsmentioning

confidence: 99%

Thyroid Hormone Metabolism in Amniotic and Allantoic Fluids of the Sheep

1975

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ExtractAmniotic and allantoic fluid samples were obtained at the time o f uterotomy from healthy pregnant ewes at various times during gestation. I n most instances maternal and fetal blood was collected simultaneously. Serum thyroxine (T, and triiodithyronine (T, ) and free T, were measured in most samples. T o determine whether conjugates o f T, were present in amniotic and allantoic fluids. T, was measured before and after 24-hr acid hydrolysis. T, and T, turnover from amniotic fluid were measured using kinetic methods after intravenous or intraamniotic fluid injection o f radiolabeled hormones.T, was unmeasurable ( < 15 ng/100 m l ) i n amniotic and allantoic fluids as well as i n most fetal serum samples. T, levels in amniotic fluid were low before 120 days o f gestation (0.17 & 0.03 Leg/ 100 ml; mean and S E M ) and increased progressively thereafter to term. The mean (and S E M ) concentration between 124 and 152 days was 0.33 =t 0.03 pg/100 ml. T, concentrations in allantoic fluid were relatively high before 120 days (0.57 0.1 pg/lOO m l ) and did not increase with progression o f the pregnancy. There were no correlations between amniotic and allantoic fluid T, concentrations, between amniotic fluid T,, and either maternal or fetal serum T, levels. or between allantoic fluid T, and maternal or fetal serum T, at any time during gestation. Mean free T, concentrations were similar i n fetal serum and amniotic fluid and higher than values in maternal serum or allantoic fluid.Mean amniotic and allantoic fluid T, concentrations by radioimmunoassay ( R I A ) increased significantly after acid hydrolvsis, suggesting that one-third to one-half the T, in these fluids is present as T, conjugate.Radiolabeled T, or T, was injected into amniotic fluid and recovery o f hormone label was measured in serum and tissues at 24 hr for comparison with recoveries after intravenous injection. Mean T, radioactivity i n several organs or tissues was nearly identical whether the hormone was injected intravenously or into amniotic fluid. I n addition, the total residual labeled T, or T, in the fetal sheep (percentage dose per fetus) at the time of killing was similar after intravenous and intra-amniotic fluid injection. Chromatograms o f butanol-ammonia extracts o f fetal sera after intraamniotic or intravenous injection o f labeled T, were similar and showed predominantly T,; a significant iodide peak was observed as well as a small peak o f radioactivity with an R, corresponding to that of T, conjugate.The results indicate that there are low but measurable concentrations o f T, in amniotic and allantoic fluids of pregnant sheep. There appears t o be minimal exchange o f hormone between allantoic and amniotic fluid. Amniotic fluid T, levels, in contrast to allantoic fluid concentrations, increase progressively during the last half o f gestation. Although free T, concentrations are similar in amniotic fluid and i n fetal serum, and are higher than i n allantoic fluid o r maternal serum, the lack of correlation between T, levels i ...

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Section: Methodsmentioning

confidence: 99%

Thyroid Hormone Metabolism in Amniotic and Allantoic Fluids of the Sheep

1975

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“…Results are expressed as the mean induction of T4-treated D1-or D2-transfected wells paired with cells transfected with an inactive D2, a COOH-terminal FLAG-tagged human D2 in which Ala replaced Glu163 (31). To prevent reentry of the T3 produced from T4 deiodination after it exited the cell, we added T3-Ab to the medium (1:1,000) (32). Luciferase activity was measured by a commercial kit (Luciferase Assay System; Promega).…”

Section: Reagentsmentioning

confidence: 99%

Type 2 iodothyronine deiodinase is the major source of plasma T3 in euthyroid humans

Maia

Kim²,

Huang³

et al. 2005

Journal of Clinical Investigation

298

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The relative roles of the types 1 and 2 iodothyronine deiodinases (D1 and D2) in extrathyroidal 3,5,3′-triiodothyronine (T 3 ) production in humans are unknown. We calculated the rate of thyroxine (T 4 ) to T 3 conversion by intact cells transiently expressing D1 or D2 at low (2 pM), normal (20 pM), and high (200 pM) free T 4 concentrations. Deiodinase activities were then assayed in cell sonicates. The ratio of T 3 production in cell sonicates (catalytic efficiency) was multiplied by the tissue activities reported in human liver (D1) and skeletal muscle (D2). From these calculations, we predict that in euthyroid humans, D2-generated T 3 is 29 nmol/d, while that of D1-generated T 3 is 15 nmol/d, from these major deiodinase-expressing tissues. The total estimated extrathyroidal T 3 production, 44 nmol/d, is in close agreement with the 40 nmol T 3 /d based on previous kinetic studies. D2-generated T 3 production accounts for approximately 71% of the peripheral T 3 production in hypothyroidism, but D1 for approximately 67% in thyrotoxic patients. We also show that the intracellular D2-generated T 3 has a greater effect on T 3 -dependent gene transcription than that from D1, which indicates that generation of nuclear T 3 is an intrinsic property of the D2 protein. We suggest that impairment of D2-generated T 3 is the major cause of the reduced T 3 production in the euthyroid sick syndrome. IntroductionThe monodeiodination of thyroxine (T 4 ) to 3,5,3′-triiodothyronine (T 3 ) activates the major secretory product of the iodine-sufficient human thyroid gland, producing approximately 80% of the circulating T 3 in humans. The types 1 and 2 iodothyronine deiodinases (D1 and D2) are members of a family of oxidoreductases that catalyze this reaction (1). These integral membrane proteins contain the rare amino acid selenocysteine (encoded by a UGA) in their active site. Both D1 and D2 require an as-yet-unidentified cofactor for the reaction. Several decades ago, D1 was identified in the liver and the kidney of rats and humans, and it is often assumed that this enzyme is the source of most of the plasma T 3 in humans. The more recent discovery of D2 mRNA and activity in human skeletal muscle suggests that D2 could also be a significant source for plasma T 3 production in humans, but this has not been quantitatively defined (1).The biochemical and molecular properties of D2 seem ideal for extrathyroidal T 3 production. Its activity is tightly controlled by the concentration of its preferred substrate, T 4 , since catalysis accelerates the ubiquitination of this enzyme, inactivating it and accelerating its degradation in proteasomes (2). The half-life of D2 in normal cells is 20-30 minutes in the presence of T 4 . The transcription of the type 2 deiodinase (DIO2) gene is also negatively regulated by T 3 (3). In contrast, the D1 protein has a long half-life (>12 hours), and the transcription of the human type 1 deiodinase (DIO1) gene is markedly stimulated by T 3 , just the opposite of what would be expected in a typical feedba...

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“…Other procedures Serum T4 and T3 concentrations were measured by previously described methods (22,23). Iodothyronine stripped rat serum instead of human serum was used for the standard curves in the radioimmunoassays.…”

Section: Studies Injecting Anti-t3 Antibodiesmentioning

confidence: 99%

Production rates and turnover of triiodothyronine in rat-developing cerebral cortex and cerebellum. Responses to hypothyroidism.

Silva¹,

Matthews²

1984

J. Clin. Invest.

109

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As bstract. Local 5'-deiodination of serum thyroxine (T4) is the main source of triiodothyronine (T3) for the brain. Since we noted in previous studies that the cerebral cortex of neonatal rats tolerated marked reductions in serum T4 without biochemical hypothyroidism, we examined the in vivo T4 and T3 metabolism in that tissue and in the cerebellum of euthyroid and hypothyroid 2-wk-old rats. We also assessed the contribution of enhanced tissue T4 to T3 conversion and decreased T3 removal from the tissues to the T3 homeostasis in hypothyroid brain. Congenital and neonatal hypothyroidism was induced by adding methimazole to the drinking water. Serum, cerebral cortex (Cx), cerebellum (Cm), liver (L) and kidney (R) concentrations of 1251-T4, '25I-T3(T4), and 13'I-T3 were measured at various times after injecting '251-T4 and 13'I-T3. The rate of T3 removal from the tissues was measured after injecting an excess of anti-T3-antibody to rats previously injected with tracer T3. In euthyroid rats, fractional turnover rates of T3 per hour were: Cx, 0.26±0.02 (SE); Cm, 0.20±0.02; L, 0.98±0.07; R, 0.97±0.12; and the calculated unidirectional plasma T3 clearance by these tissues were, in milliliters per gram per hour: Cx = 0.38, Cm = 0.32, L = 5.0, and R = 5.6. In hypothyroidism, the fractional removal rates and clearances were reduced in all tissues, in cortex and cerebellum by 70%, and in liver and kidney ranging from 30 to 50%. While >80% May 24, 1984. of the '25I-T3(T4) in the brain tissues of euthyroid rats was locally produced, in hypothyroid cerebral cortex and cerebellum the integrated concentrations of 1251. T3(T4) were 2.7-and 1.5-fold greater than in euthyroid rats. In the Cx, this response resulted from an approximately sixfold increase in fractional conversion and an approximately fourfold decrease in T3 removal rate hampered by a decreased uptake of T4 from plasma, whereas in Cm the response resulted only from the reduced T3 removal rate. In euthyroid rats, the calculated production rate of T3 in nanograms per gram per hour by the Cx was 0.96 and 0.89 by the Cm, which on a per organ basis equals 15 and 2%, respectively, of the extrathyroidal production rate as assessed in the body pool exchanging with plasma. Several conclusions can be drawn: (a) Production of T3 by developing brain is a very active process in agreement with the need of thyroid hormones during this period. (b) The brainplasma exchange of T3 is slow compared with that of L or R. (c) This, along with the active local production, explains the predominant role of the latter as a source of T3 for the brain. (d) In hypothyroidism, the Cx is protected by an increase in the efficiency of T4 to T3 conversion and a prolonged residence time of T3 in the tissue, whereas the Cm is protected only by the latter. Because of the large fraction of the tissue T3 produced locally and the active turnover rate of T3 in the brain, reductions in T3 removal rate are of utmost importance for T3 homeostasis in these tissues.

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Direct immunoassay of triiodothyronine in human serum

Cited by 209 publications

References 19 publications

Thyroid Hormone Metabolism in Amniotic and Allantoic Fluids of the Sheep

Thyroid Hormone Metabolism in Amniotic and Allantoic Fluids of the Sheep

Type 2 iodothyronine deiodinase is the major source of plasma T3 in euthyroid humans

Production rates and turnover of triiodothyronine in rat-developing cerebral cortex and cerebellum. Responses to hypothyroidism.

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