Analytical performance evaluation of thyroid-stimulating hormone receptor antibody (TRAb) immunoassays.

BACKGROUND: Thyroid-stimulating hormone receptor (TSHR)-activating autoantibodies stimulate thyroid growth and hormone synthesis/secretion, causing hyperthyroidism of Graves' disease (GD). TRAb measurement helps diagnose GD and is an important first test in evaluating hyperthyroidism according to the recent American Thyroid Association guidelines. We compared the performance of the BRAHMS TRAK Kryptor (Thermo Scientific) and Roche cobas TRAb immunoassays for use in GD. METHOD: Method comparison (n = 40) and clinical agreement were assessed between the Kryptor, cobas e411, and cobas e601. The analytical performance of Kryptor and cobas e411 were assessed for within- and between-day imprecision across 20 days, linearity, functional assay sensitivity (FAS), dilution recovery, and cut-off verification. RESULTS: The Kryptor, e411, and e601 TRAb immunoassays correlated well (r > 0.95, overall percent agreement = 0.95, Cohen's kappa = 0.90). With a total allowable error of 20%, percent bias was within 13%, which was minimally negative at <20 IU/L, but highly positive (33%-34%) >20 IU/L. The Kryptor, but not e411, was linear across the claimed analytical measuring range (AMR). The claimed functional assay sensitivity (FAS), which was close to the clinical GD cut-off 1.8 IU/L, was verified for Kryptor and e411. CONCLUSION: Overall, our evaluation demonstrates acceptable comparability between TRAb immunoassays with in-house imprecision up to 13% and 10% on Kryptor and e411, respectively. While Roche has preferable calibration frequency and on-board reagent stability, both platforms demonstrate acceptable imprecision using patient samples at their claimed FAS, which is important for GD diagnosis. Diluted results (using a negative patient pool as diluent) exhibits proportional positive bias on the Kryptor relative to the Roche methods.

Absence of an effect of dietary corn oil content on plasma prolactin, progesterone, and 17 beta-estradiol in female Sprague-Dawley rats.

Enhancement of mammary tumorigenesis in rats by high-fat diets has been postulated to be due to altered hormonal status. Elevated serum prolactin and, in some cases, estrogen have been reported in rats fed diets high in corn oil or lard that increase 7,12-dimethylbenz(a)anthracene (DMBA) tumorigenesis. However, we have found no difference in plasma prolactin during the proestrus surge or at the other stages of the estrous cycle in rats fed a diet high (24%) in corn oil that augments DMBA-induced tumorigenesis or a control diet containing 5% corn oil. There was no effect of the same dietary treatment on plasma progesterone or serum estradiol in the same experiments. In addition, we found that DMBA administration did not change the blood concentration of any of the three hormones. Female Sprague-Dawley rats, bearing atrial cannulae and given a carcinogenic dose of DMBA at 8 weeks of age, were studied 2 to 5 weeks or 10 to 13 weeks after DMBA administration. Rats given only the oil vehicle for DMBA were studied at the same ages. Blood samples were taken from rats with 4-day estrous cycles at 3-hr intervals on the day of proestrus and at 6- to 12-hr intervals on the other days of the cycle. No effect of dietary corn oil content or of DMBA administration on plasma prolactin and progesterone was detected in either age group. On the afternoon of proestrus, rats were sampled more frequently to examine the hormonal patterns in detail; again, no dietary effect was detected. Serum estradiol was measured in rats that were fed the control or high-corn-oil diets, treated with DMBA or vehicle, and decapitated on one of the 4 days of the estrous cycle. There was no detectable effect of dietary corn oil content or DMBA treatment during any stage of the cycle. All three hormones showed qualitative and quantitative patterns identical to normal cycling rats at both ages studied. Our results indicate that earlier reports of an effect of dietary fat on blood prolactin and estrogen content may have been due either to diets low in essential fatty acids or to anesthesia-induced hormonal responses.

Photodynamic effect in an experimental bladder tumor treated with intratumor injection of hematoporphyrin derivative.

Photodynamic therapy (PDT) is an experimental treatment modality for malignant tumors. It is based on the principle that a photosensitizer, such as hematoporphyrin derivative (HPD), is retained in higher concentrations in tumors than in surrounding nonmalignant tissues and that photoactivation of the sensitizer can be used to evoke tumor destruction. However, retention of the systemic injection of HPD is not limited to malignant tissues. This lack of specific tumor localization thus reduces the therapeutic ratio of the treatment and causes skin photosensitivity and possible systemic toxicity. Injection of HPD directly into the tumor, on the other hand, has been shown to yield higher levels of the drug in the tumor and lower levels in normal tissues, in comparison with systemic administration. In this study, we examined the photodynamic effect on s.c. implanted mouse bladder tumors subjected to intratumor (i.t.) and i.p. HPD injections. Tumor cell killing, measured by cell survival, was observed in both the it. and i.p. groups and was dependent on fluence and HPD dosage. However, no significant enhancement of cell killing was observed in the i.t. injected tumors, despite the higher porphyrin levels in these tumors. Histological examination of the effect of PDT on the blood vessels indicated that while cell death accompanied severe hemorrhage in the i.p. injected tumors, in the i.t. tumors there was much less hemorrhage and intact blood vessels remained. This observation suggests that with i.t. administration, direct photodynamic action may play a significant role in the tumor cell killing, in contrast to systemic administration, in which destruction of the blood vessels is believed to be the main cause of tumor destruction.

Permanently charged chiral 1,4-dihydropyridines: molecular probes of L-type calcium channels. Synthesis and pharmacological characterization of methyl(omega-trimethylalkylammonium) 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate iodide, calcium channel antagonists.

We report the synthesis of the single enantiomers of permanently charged dihydropyridine derivatives (DHPs with alkyl linker lengths of two and eight carbon atoms) and their activities on cardiac and neuronal L-type calcium channels. Permanently charged chiral 1,4-dihydropyridines and methyl (omega)-trimethylalkylammonium) 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate iodides were synthesized in high optical purities from (R)-(-) and (S)-(+)-1,4-dihydro-2,6-dimethyl-5-methoxycarbonyl-4-(3-nitrophenyl)-3-+ ++pyridinecarboxylic acid, obtained by resolution of racemic 1,4-dihydro-2,6-dimethyl-5-methoxycarbonyl-4-(3-nitrophenyl)-3-pyridi necarboxylic acid. Competition binding experiments with radioligand [3H]-(+)-PN200-110 and the block of whole cell barium currents through L-type calcium channels in GH4C1 cells show that the compounds with the eight-carbon alkyl linker optimally block the L-type Ca2+ channels, and that the S-enantiomer is more potent than the R-enantiomer.

Crystal structures and pharmacologic activities of 1,4-dihydropyridine calcium channel antagonists of the isobutyl methyl 2,6-dimethyl-4-(substituted phenyl)-1,4-dihydropyridine-3,5-dicarboxylate (nisoldipine) series.

A series of isobutyl methyl 2,6-dimethyl-4-(X-substituted phenyl)-1,4-dihydropyridine-3,5-dicarboxylates (X = H, 2-NO2, 3-NO2, 3-CN, 3-MeO, 4-F, 2-CF3, 3-CF3, and 4-Cl) related to and including nisoldipine (X = 2-NO2) has been synthesized, their solid-state structures determined by X-ray analysis (X = H, 2-NO2, 3-NO2, 3-CN, 3-MeO, and 4-F), and their pharmacologic activities determined, as the racemic compounds, against [3H]nitrendipine binding and K+-depolarization-induced tension responses in intestinal smooth muscle as measures of Ca2+ channel antagonist activity. Comparisons of structure are presented to previously analyzed 1,4-dihydropyridines. The degree of 1,4-dihydropyridine ring puckering is dependent on the nature and position of the phenyl ring substituent and the adopted interring conformation. Different ester substituents affect 1,4-dihydropyridine ring puckering to a small extent in most cases. Pharmacologic and radioligand binding activities for the nine compounds studied show a parallel dependence on phenyl ring substituent, but the compounds are approximately 10-fold more active in the radioligand binding assay than in the pharmacologic assay. Consistent with a previous report for the nifedipine series (Fossheim et al. J. Med. Chem. 1982, 25, 126), pharmacologic activity increases with increasing 1,4-dihydropyridine ring planarity.

Lipophilic 4-isoxazolyl-1,4-dihydropyridines: synthesis and structure-activity relationships.

A series of 4-isoxazolyl-1,4-dihydropyridines bearing lipophilic side chains at the C-5 position of the isoxazole ring have been prepared. The calcium channel antagonistic activity of these compounds has been evaluated. A hypothetical model for binding of these compounds in the calcium channel is proposed, and the validity of this model is evaluated based on the SAR of this series of calcium binding, especially for the two most active derivatives, 1a, g. The solid-state structure for the most active compound, 1a, has also been determined, and its important features are reported.

Calcium-dependence and antagonism of responses to alpha 1- and alpha 2-adrenoceptor agonists in vascular tissues from hypertensive and normotensive rats.

Inhibition by D600 (methoxyverapamil) of responses to an alpha 2-adrenoceptor selective agonist, B-HT 920, (6-allyl-2-amino-5, 6, 7, 8-tetrahydro-4H-thiazolo [4, 5-d] azepin dihydrochloride), an alpha 1-adrenoceptor selective agonist, phenylephrine (PE), and a nonselective agonist, noradrenaline (NA), was studied in isolated preparations of the aortae and carotid arteries obtained from young (5-7 weeks) and old (15-17 weeks) hypertensive (SHR) and normotensive (WKY) rats. Maximum responses of WKY tissues to B-HT 920 were the most sensitive, PE-induced responses the least sensitive and maximum responses to NA were intermediate in their sensitivity to inhibition by D600. Sub-maximal responses to NA and PE were not different in their sensitivity to inhibition by D600, but were less sensitive than the responses to B-HT 20. Sub-maximal responses to PE were significantly more sensitive to D600 inhibition than were the maximal responses to this agonist. NA-induced responses of tissues from older SHR were less sensitive to inhibition by D600 when compared to responses in WKY rats. Responses to B-HT 920, in tissues suspended in calcium-free solutions, showed the largest decline compared to NA- and PE-induced responses. We conclude that responses to B-HT 920 largely utilize extracellular calcium. PE- and NA- induced responses mobilize extracellular calcium to varying degrees depending upon the concentration of the agonist employed to elicit the response.

Dietary sodium intake: influence on calcium channels and urinary calcium excretion in spontaneously hypertensive rats.

Binding of the 1,4-dihydropyridine [3H]PN200 110 was employed as an index of cardiac Ca2+ channels in normotensive (WKY) and spontaneously hypertensive (SHR) rats during 4 weeks of normal (0.73% NaCl) and high (8% NaCl) sodium diets when the rats were between 20 and 24 weeks of age. Binding site density was not different at the beginning of the study but was increased significantly (P less than 0.01) after 1 week in the SHR on a high sodium diet; this difference was not apparent at 2, 3 or 4 weeks of the diet. During this same period, the urinary Ca2+ excretion in SHR was enhanced significantly (P less than 0.01) and the urinary calcium/sodium ratio was elevated during the high sodium intake period.

The actions of peppermint oil and menthol on calcium channel dependent processes in intestinal, neuronal and cardiac preparations.

The activities of menthol and peppermint oil were determined in guinea-pig ileal smooth muscle, in rat and guinea-pig atrial and papillary muscle, in rat brain synaptosomes and in chick retinal neurones by pharmacological 45Ca2+ uptake and radioligand binding assays. Menthol is a major constituent of peppermint oil and is approximately twice as potent as peppermint oil as an inhibitor of K+ depolarization-induced and electrically stimulated responses in ileum and electrically stimulated atrial and papillary muscles. IC50 values in the ileal preparation ranged from 7.7 to 28.1 micrograms ml-1 and in the cardiac preparations from 10.1 to 68.5 micrograms ml-1. Similar potencies were demonstrated against K+ depolarization-induced 45Ca2+ uptake in synaptosomes and against K+ depolarization and Bay K 8644-induced uptake in chick retinal neurons. IC50 values for menthol inhibition of K+ and Bay K 8644 responses in the retinal neurons were 1.1 x 10(-4) M (17.2 micrograms ml-1) and 1.75 x 10(-4) M (26.6 micrograms ml-1), respectively, and for peppermint oil were 20.3 and 41.7 micrograms ml-1 respectively. Both menthol and peppermint oil inhibited specific [3H]nitrendipine and [3H]PN 200-110 binding to smooth and cardiac muscle and neuronal preparations with potencies comparable to, but slightly lower than, those measured in the pharmacological and 45Ca2+ uptake experiments. Binding of menthol and peppermint oil, studied at 78 micrograms ml-1, was competitive against [3H]nitrendipine in both smooth muscle and synaptosome preparations. The data indicate that both menthol and peppermint oil exert Ca2+ channel blocking properties which may underlie their use in irritable bowel syndrome. Ca2+ channel antagonism may not be the only pharmacological effect of menthol and peppermint oil contributing to intestinal smooth muscle relaxation.

Short-term regulation of neuronal calcium channels by depolarization.

The 1,4-dihydropyridine-sensitive voltage-gated Ca2+ channel is widely distributed in excitable cells. The channel and its several associated drug binding sites are known to be up- and downregulated by a variety of homologous and heterologous influences including membrane depolarization. The neurosecretory GH4C1 cell line possesses L-type channels. Depolarization of these cells by elevated K+ increases the binding affinity of 1,4-dihydropyridines and decreases the number of 1,4-dihydropyridine binding sites and functional channels. There is a coordinate upregulation of the number of muscarinic receptors. This membrane potential- and Ca(2+)-calmodulin-dependent process of channel downregulation may involve internalization of the channel heteromeric complex or, more plausibly, a dissociation of the complex and a concomitant loss of both binding and permeation functions.

The binding interactions of Ro 40-5967 at the L-type Ca2+ channel in cardiac tissue.

Ro 40-5967 [(1S,2S)-2-[2[3-(2-benzamidopropyl]- methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphthyl- methoxyacetate] is a new Ca2+ channel antagonist active at L-type channels. Radioligand binding studies in cardiac tissue show that Ro 40-5967 does not inhibit 1,4-dihydropyridine binding, but does inhibit diltiazem, desmethoxyverapamil and SR 33557 binding with IC50 values of 8 x 10(-9), 10(-8) and 5 x 10(-8) M, respectively. Equilibrium and kinetic binding studies showed that Ro 40-5967 inhibited both desmethoxyverapamil and SR 33557 binding in an apparently competitive manner. Ro 40-5967 defines an additional and possibly unique antagonist binding site on the L-type voltage-gated Ca2+ channel.

Interactions of analogs of the 1,4-dihydropyridine tiamdipine in cardiac and smooth muscle.

Two series of 1,4-dihydropyridines related to tiamdipine, 2-(2-aminoethylthio)methyl-3-carboethoxy-5-carbomethoxy-6- methyl-4-(3-nitrophenyl)-1,4-dihydropyridine, have been evaluated for their pharmacologic and radioligand binding properties in smooth and cardiac muscle. In the tiamdipine series the influence of phenyl ring substitution, 3-Cl, 3-MeO and 3-CF3, was greatly reduced relative to the N-formyl and neutral nifedipine derivatives. Consistent with our previous observations onset and offset of action were greatly reduced by the presence of the amine side chain. In tiamdipine analogs also bearing an asymmetric substituent at C-2, chirality at C-4 was determinant for activity.

Effect of an homologous series of aliphatic alcohols on neuronal and smooth muscle voltage-dependent Ca2+ channels.

The acute inhibitory actions of alcohol on K(+)-stimulated 45Ca2+ uptake into synaptosomes shows regional variation in sensitivity throughout the brain, suggesting the possibility of a selective action on a specific Ca2+ channel subtype. This was examined by comparing the effects of a homologous series of aliphatic alcohols on synaptosomal Ca2+ channels with their actions on K(+)-stimulated Ca2+ channels in guinea-pig intestinal longitudinal muscle, which have been demonstrated to be of the L-type. K(+)-stimulated contraction of and [3H]nitrendipine binding to smooth muscle were both inhibited by the alcohols at similar concentrations, with the potency increasing with chain length. In synaptosomes, however, K(+)-stimulated 45Ca2+ uptake was 5-30 times more sensitive to the inhibitory actions of alcohol than were [3H]nitrendipine and [125I]omega-conotoxin binding. These observations suggest that K(+)-stimulated 45Ca2+ uptake is mediated by a non-L non-N type channel which is more sensitive to the acute effects of alcohols. This is supported by the observation that K(+)-stimulated 45Ca2+ uptake which is insensitive to L- and N-channel antagonists was inhibited by funnel web spider venom.

Effect of thyroid status on beta-adrenoceptors and calcium channels in rat cardiac and vascular tissue.

To determine the influence of thyroid hormone on beta-adrenoceptors and Ca2+ channels, rats were treated with thyroxine (75 micrograms/100 g sc daily for 5 days) or propylthiouracil (0.05% in drinking water for 30 days). beta-Adrenoceptor density in ventricular tissue, measured by [125I]iodocyanopindolol binding, was significantly increased and decreased respectively, following thyroxine or propylthiouracil treatment to 124.7 +/- 7.11 fmol/mg protein and 71.98 +/- 5.37 fmol/mg protein from euthyroid (control) levels of 93.7 +/- 4.58 fmol/mg protein. Ca2+ channel density, measured by [3H]nitrendipine binding, was altered in the opposite direction; it was significantly decreased and increased to 324 +/- 24 fmol/mg protein and 691 +/- 31 fmol/mg protein from 562 +/- 35 fmol/mg protein after thyroxine or propylthiouracil treatment, respectively. No changes in affinity of either ligand were observed. Responses of isolated papillary muscles from propylthiouracil-treated animals accorded with changes seen in the binding studies. The geometric mean EC50 of isoproterenol increased from 9.5 x 10(-9) mol/l to 5.5 x 10(-8) mol/l, and the EC50 for calcium decreased from 3.16 x 10(-3) mol/l to 1.36 x 10(-3) mol/l; moreover, the responsiveness to the Ca2+ channel activator Bay K 8644 was increased. The corresponding responses in thyroxine-treated animals could not be examined because of prominent arrhythmic activity. As with papillary muscles the sensitivity of left atria to isoproterenol was decreased after treatment with propylthiouracil, with geometric mean EC50 values increasing from 3.21 x 10(-9) mol/l to 89.4 x 10(-9) mol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Basic fibroblast growth factor (FGF-2) affects development of acoustico-vestibular neurons in the chick embryo brain in vitro.

The effects of basic fibroblast growth factor (FGF-2) on presumptive auditory and vestibular neurons from the medulla were studied in primary cell cultures. The part of the rhombic lip that forms nucleus magnocellularis (homologue of the mammalian anteroventral cochlear nucleus) was explanted from white leghorn chicken embryos at Hamburger-Hamilton stage 28 (E5.5), the time when precursors of the magnocellularis bushy cells migrate and begin to differentiate in situ. In vitro the neuroblasts migrated onto 2-D substrates of purified collagen, differentiated, and expressed neuronal markers. One-half of the cultures were supplemented with human recombinant FGF-2 (10 ng/ml daily) for 5-7 days; the others, with fetal bovine serum. FGF-2 more than doubled the length of neurite outgrowth during the first 3 day treatment compared to serum, but the number of migrating neuroblasts was unaffected. Although neurites attained greater lengths in FGF-2, they usually degenerated after 4-5 days; in serum their growth continued for several weeks. Differentiation of neuronal structure, including axons and dendrites, began within 1-2 days in bFGF but required at least 5-7 days in serum. Histochemical observations in vitro and in situ with antibodies to FGF receptor demonstrated immunopositive patches on acoustico-vestibular neuroblasts at stage 28, when they are migrating and first forming their axons. The findings suggest that FGF-2 stimulates neurite outgrowth in the cochlear and vestibular nuclei. FGF-2 may accelerate cell death by overstimulating neuroblasts, but other factors are needed to sustain their further development.

Basic fibroblast growth factor (FGF-2) affects development of acoustico-vestibular neurons in the chick embryo brain in vitro.

The effects of basic fibroblast growth factor (FGF-2) on presumptive auditory and vestibular neurons from the medulla were studied in primary cell cultures. The part of the rhombic lip that forms nucleus magnocellularis (homologue of the mammalian anteroventral cochlear nucleus) was explanted from white leghorn chicken embryos at Hamburger-Hamilton stage 28 (E5.5), the time when precursors of the magnocellularis bushy cells migrate and begin to differentiate in situ. In vitro the neuroblasts migrated onto 2-D substrates of purified collagen, differentiated, and expressed neuronal markers. One-half of the cultures were supplemented with human recombinant FGF-2 (10 ng/ml daily) for 5-7 days; the others, with fetal bovine serum. FGF-2 more than doubled the length of neurite outgrowth during the first 3 day treatment compared to serum, but the number of migrating neuroblasts was unaffected. Although neurites attained greater lengths in FGF-2, they usually degenerated after 4-5 days; in serum their growth continued for several weeks. Differentiation of neuronal structure, including axons and dendrites, began within 1-2 days in bFGF but required at least 5-7 days in serum. Histochemical observations in vitro and in situ with antibodies to FGF receptor demonstrated immunopositive patches on acoustico-vestibular neuroblasts at stage 28, when they are migrating and first forming their axons. The findings suggest that FGF-2 stimulates neurite outgrowth in the cochlear and vestibular nuclei. FGF-2 may accelerate cell death by overstimulating neuroblasts, but other factors are needed to sustain their further development.