A Homogeneous Time-Resolved Fluorescence Immunoassay Method for the Measurement of Compound W.

OBJECTIVE: Using compound W (a 3,3'-diiodothyronine sulfate [T2S] immuno-crossreactive material)-specific polyclonal antibodies and homogeneous time-resolved fluorescence immunoassay assay techniques (AlphaLISA) to establish an indirect competitive compound W (ICW) quantitative detection method. METHOD: Photosensitive particles (donor beads) coated with compound W or T2S and rabbit anti-W antibody were incubated with biotinylated goat anti-rabbit antibody. This constitutes a detection system with streptavidin-coated acceptor particle. We have optimized the test conditions and evaluated the detection performance. RESULTS: The sensitivity of the method was 5 pg/mL, and the detection range was 5 to 10 000 pg/mL. The intra-assay coefficient of variation averages <10% with stable reproducibility. CONCLUSIONS: The ICW-AlphaLISA shows good stability and high sensitivity and can measure a wide range of compound W levels in extracts of maternal serum samples. This may have clinical application to screen congenital hypothyroidism in utero.

Steric and electronic factor comparisons in hydrotris(3-phenylpyrazolyl)borate nickel(II) aryloxides.

Hydrotris(pyrazolyl)borate (Tp) ligands, also known as scorpionates, are potent tridentate donors that effectively bind metal ions in a face-capping array. Hydrotris(3-phenylpyrazolyl)borate enforces a tetrahedral environment on NiII to model metalloenzymes. The syntheses and structural characterizations of a number of [hydrotris(3-phenylpyrazolyl)borato]nickel(II) aryloxides were performed to provide insight into the environment of the model active site; these compounds are chlorido[hydrotris(3-phenylpyrazolyl-κN2)borato](3-phenyl-1H-pyrazole-κN2)nickel(II) chloroform monosolvate, [Ni(C27H22BN6)Cl(C9H8N2)]·CHCl3, (2), [hydrotris(3-phenylpyrazolyl-κN2)borato](phenolato-κO)nickel(II), [Ni(C27H22BN6)(C6H5O)], (3), (2,6-dimethylphenolato-κO)[hydrotris(3-phenylpyrazolyl-κN2)borato]nickel(II) [Ni(C27H22BN6)(C8H9O)], (4), (4-tert-butylphenolato-κO)[hydrotris(3-phenylpyrazolyl-κN2)borato]nickel(II), [Ni(C27H22BN6)(C10H13O)], (5), and [hydrotris(3-phenylpyrazolyl-κN2)borato](phenolato-κO)(tetrahydrofuran-κO)nickel(II) tetrahydrofuran monosolvate, [Ni(C27H22BN6)(C6H5O)(C4H8O)]·C4H8O, (6). Alkyl groups, e.g. tert-butyl in (5) and methyl in (4), electronically activate the aryloxide group to intramolecular π-π stacking but can be frustrated by steric encumbrance at the interacting ring faces. The flexibility at the nickel coordination site, afforded by the uncrowded B atom of the TpPh ligand, allows tetrahydrofuran coordination in the phenolate compound.

3,3'-Diiodothyronine sulfate cross-reactive material (compound W) in human newborns.

BACKGROUND: Thyrosulfoconjugation appears to facilitate fetal-to-maternal transfer of 3,3'-diiodothyronine-sulfate (T(2)S). Elevated maternal levels of T(2)S cross-reactive material (compound W) are found in humans, with higher levels found in venous cord blood than in arterial samples. These findings are consistent with the postulate that the placenta plays an essential role in compound W production. METHODS: Serum compound W levels were measured by a T(2)S-specific radioimmunoassay in 60 serum samples from newborns with hyperbilirubinemia, age 1-30 d. In addition, 59 maternal serum samples, from day 1 to day 7 after uneventful deliveries, were studied. RESULTS: As compared with day 1, at day 5, the mean (±SE) compound W level fell to 43.5 ± 6.8% (decay half-life (t(1/2)) = 4.12 d) and to 33.7 ± 4.6% (decay t(1/2) = 2.82 d) in the newborn and maternal groups, respectively. In the mothers, the level continued to decline along the same slope through day 7. In the newborns, however, the mean compound W level entered a slower phase of decay after the fifth day with a decay t(1/2) = 10.9 d. CONCLUSION: Compound W is cleared at similar rates in newborn and postpartum maternal sera. This is consistent with the postulate that compound W is produced in the placenta.

Fetal-to-maternal transfer of thyroid hormone metabolites in late gestation in sheep.

3,3'-Diiodothyronine sulfate (T2S) derived from T3 of fetal origin is transferred to the maternal circulation and contributes significantly to the maternal urinary pool. The present study quantitatively assesses the fetal to maternal transfer of T4 metabolites compared with those of T3. Labeled T4 or T3 was infused intravenously to four singleton fetuses in utero in each group at gestational age 138 +/- 3 d. Maternal and fetal serum and maternal urine samples were collected hourly for 4 h and at 24 h (serum) or in pooled 4-24 h samples (urine). Radioactive metabolites were identified by HPLC and by specific antibody in serum and urine extracts and expressed as percentage infusion dose per liter. The results demonstrate a rapid clearance of labeled T3 from fetal serum (disappearance T(1/2) of 0.7 h versus 2.4 h for T4 in the first 4 h). The metabolites found in fetal serum after labeled T3 infusion were T2S > T3 > T3S; in maternal urine, T2S > unconjugated iodothyronines (UI) > T3S > unknown metabolite (UM). After labeled T4 infusion, the metabolites in fetal serum were rT3 > T3 > T2S > T4S in the first 4 h, and rT3 = T3 = T4S = T2S > T3S at 24 h; in maternal urine we found T2S > UM > UI > T4S > T3S in the first 4 h and UM > T2S > UI in 4-24 h pooled sample. In conclusion, the conversion of T3 to T2S followed by fetal to maternal transfer of T2S and other iodothyronines appears to contribute importantly to maintaining low fetal T3 levels in late gestation.

Alternate pathways of thyroid hormone metabolism.

The major thyroid hormone (TH) secreted by the thyroid gland is thyroxine (T(4)). Triiodothyronine (T(3)), formed chiefly by deiodination of T(4), is the active hormone at the nuclear receptor, and it is generally accepted that deiodination is the major pathway regulating T(3) bioavailability in mammalian tissues. The alternate pathways, sulfation and glucuronidation of the phenolic hydroxyl group of iodothyronines, the oxidative deamination and decarboxylation of the alanine side chain to form iodothyroacetic acids, and ether link cleavage provide additional mechanisms for regulating the supply of active hormone. Sulfation may play a general role in regulation of iodothyronine metabolism, since sulfation of T(4) and T(3) markedly accelerates deiodination to the inactive metabolites, reverse triiodothyronine (rT(3)) and T(2). Sulfoconjugation is prominent during intrauterine development, particularly in the precocial species in the last trimester including humans and sheep, where it may serve both to regulate the supply of T(3), via sulfation followed by deiodination, and to facilitate maternal-fetal exchange of sulfated iodothyronines (e.g., 3,3'-diiodothyronine sulfate [T(2)S]). The resulting low serum T(3) may be important for normal fetal development in the late gestation. The possibility that T(2)S or its derivative, transferred from the fetus and appearing in maternal serum or urine, can serve as a marker of fetal thyroid function is being studied. Glucuronidation of TH often precedes biliary-fecal excretion of hormone. In rats, stimulation of glucuronidation by various drugs and toxins may lead to lower T(4) and T(3) levels, provocation of thyrotropin (TSH) secretion, and goiter. In man, drug induced stimulation of glucuronidation is limited to T(4), and does not usually compromise normal thyroid function. However, in hypothyroid subjects, higher doses of TH may be required to maintain euthyroidism when these drugs are given. In addition, glucuronidates and sulfated iodothyronines can be hydrolyzed to their precursors in gastrointestinal tract and various tissues. Thus, these conjugates can serve as a reservoir for biologically active iodothyronines (e.g., T(4), T(3), or T(2)). The acetic acid derivatives of T(4), tetrac and triac, are minor products in normal thyroid physiology. However, triac has a different pattern of receptor affinity than T(3), binding preferentially to the beta receptor. This makes it useful in the treatment of the syndrome of resistance to thyroid hormone action, where the typical mutation affects only the beta receptor. Thus, adequate binding to certain mutated beta receptors can be achieved without excessive stimulation of alpha receptors, which predominate in the heart. Ether link cleavage of TH is also a minor pathway in normal subjects. However, this pathway may become important during infections, when augmented TH breakdown by ether-link cleavage (ELC) may assist in bactericidal activity. There is a recent claim that decarboxylated derivates of thyronines, that is, monoiodothyronamine (T(1)am) and thyronamine (T(0)am), may be biologically important and have actions different from those of TH. Further information on these interesting derivatives is awaited.

New questions regarding bioequivalence of levothyroxine preparations: a clinician's response.

A recent decision by the Food and Drug Administration (FDA) to declare various brands of levothyroxine bioequivalent has provoked objections from several physicians' organizations. These organizations assert that the method of testing bioequivalence is flawed, and that indiscriminate switching among preparations could lead to serious instances of undertreatment and overtreatment of hypothyroid patients. In this review we first list common indications for thyroid hormone administration, distinguishing its use as replacement therapy in hypothyroidism from its use to suppress thyrotropin (TSH) secretion in cases of thyroid cancer, nodules, and goiter. The dangers associated with changing to a preparation with different bioavailability are summarized, noting the particular danger of giving a more active preparation to a patient receiving TSH-suppressive doses of levothyroxine. However, these dangers are part of a larger problem: there are data showing that large numbers of patients are already receiving an improper dosage of levothyroxine, as judged from measurements of serum TSH. The recent history of FDA actions concerning levothyroxine bioequivalence and the arguments of those in disagreement are summarized. The immediate response to these problems should be better education of both patients and physicians. It is also recommended that there be further discussion of the problems in determining bioequivalence, and that consideration be given to more accurate and clinically relevant methods. Such methods should include assessment of the changes in TSH induced by each preparation in athyrotic patients.