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.

Measures of bone mineral content in mature dairy cows.

The objectives of this investigation were to assess the relationship between chemical measures and imaging estimates (radiographic photometry and dual-energy x-ray absorptiometry) of bone mineral content in dairy cows and to evaluate the effects of parity, stage of lactation, and site of measurement (fused third and fourth metacarpal bone vs. caudal vertebrae 14 and 15) on bone mineral content. In a preliminary study, the caudal vertebrae were excised from 33 cows following slaughter. Samples were analyzed by radiographic photometry and then analyzed for mineral content chemically. In a second experiment, the caudal vertebrae and right front metacarpal (sample pairs) were excised from 107 Holstein cull cows following slaughter. Parity and days in milk (DIM) of the donor animals were obtained for 43 pairs of samples. Samples were grouped by parity (1, 2, 3, and >or=4) stage of lactation (Stage 1: <90 DIM, Stage 2: 90 to 150 DIM, Stage 3: 151 to 250 DIM, and Stage 4: >250 DIM). Samples were analyzed by radiographic photometry and dual-energy x-ray absorptiometry and then analyzed for mineral content chemically. In both experiments, the relationship between mineral content estimated via the imaging techniques and mineral content measured chemically was poor, likely because of the relative maturity of animals in the sample set and lack of variation in mineral content. Ash content was higher in the metacarpal than in the caudal vertebrae, as were concentrations of Mg (expressed as a proportion of bone ash). No effects of stage of lactation were observed on bone mineral in the caudal vertebrae, but in the metacarpal, P content (proportion of total mineral) was highest in second lactation cows. Total bone mineral content (ash) was not affected by parity in the metacarpal or caudal vertebra, but Ca and P content of the metacarpal increased with parity. Noninvasive imaging techniques are not sufficiently sensitive to detect changes in mineral content or composition of mature cows, and only modest changes in bone mineral were observed with stage of lactation and parity.

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.

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.

Critical periods of basic fibroblast growth factor and brain-derived neurotrophic factor in the development of the chicken cochleovestibular ganglion in vitro.

The temporal roles of brain-derived neurotrophic factor (BDNF) and fibroblast growth factor-2 (FGF-2) in the development of sensory neurons have been studied in a cell culture preparation which models normal embryonic inner ear development (normocytic). Previous studies showed that FGF-2 stimulated migration and differentiation of ganglion cells for the first 2 days in vitro, but after 5 days led to degeneration, implicating other factors in their later development. To see if BDNF could be such a factor, otocysts were explanted from white leghorn embryos at the time when ganglion cell precursors normally start migrating from the otic epithelium. Cultures were grown in a defined medium, either with or without human recombinant FGF-2 for 2 days or with BDNF. On Day 3, FGF-2 was replaced either with BDNF in defined medium or with defined medium only. Measurements of neuroblast migration and neurite outgrowth were made by time-lapse imaging in living cultures. In cultures receiving BDNF on Day 3, cell migration and neurite outgrowth from the explant increased for more than 3 weeks but not in cultures receiving only defined medium from Day 3. Cultures did not survive more than 3-4 days when receiving either BDNF in defined medium or defined medium alone from the first day. A neutralizing antibody to BDNF inhibited neuronal migration and neurite outgrowth, and it also blocked the effects of exogenous BDNF. BDNF did not enhance the effects of FGF-2 by interacting with it. These experiments defined a temporal sequence in which FGF-2 acts early in development, while BDNF affects a later stage.

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.

Basic fibroblast growth factor affects neuronal migration and differentiation in normotypic cell cultures from the cochleovestibular ganglion of the chick embryo.

To study the role of basic fibroblast growth factor (FGF-2) in the development of sensory neurons, the cochleovestibular ganglion of the chicken embryo provides a well-characterized structure. This permits use of morphological markers in a cell culture preparation comparable to the normal embryo (normocytic). Otocysts were explanted from white leghorn embryos at Hamburger-Hamilton Stages 14-16, when ganglion cell precursors normally start migrating from the otic epithelium. The cultures were supplemented with either fetal bovine serum or human recombinant FGF-2 (in defined medium or serum) for 2 or 5 days. FGF-2 increased explant growth, neuroblast migration, and neurite outgrowth 2- to 10-fold in the first 2 days. Neuronal morphology appeared within 2-3 days with FGF-2 but required at least 4-5 days with serum. FGF-2 in defined medium stimulated early migration and differentiation, but without serum led to degeneration after 5 days. In serum, growth was later and slower but continued for at least 3 weeks. When explants were cultured in serum with a neutralizing antibody to FGF-2, but no FGF added, neuroblast migration and elongation were decreased by 2- to 4-fold, compared to serum alone. Immunocytochemistry demonstrated FGF receptor sites on the migrating ganglionic neuroblasts, on their processes and growth cones, and in the incipient ganglion and otic epithelium at Stages 15-17, both in the embryo and in vitro. The findings suggest that FGF-2 stimulates early migration and differentiation of ganglion cells by activating the receptors of neuroblasts or their precursors in the embryonic otocyst. However, other factors must sustain their later development.

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.

L-type calcium channels: asymmetrical intramembrane binding domain revealed by variable length, permanently charged 1,4-dihydropyridines.

We have used an homologous series of dihydropyridine (DHP) derivatives to determine the location of the binding domain for DHPs on cardiac L-type calcium channels, relative to the extracellular and intracellular membrane surfaces. The series of test molecules consisted of DHP analogs in which the DHP moiety was linked to either a neutral (-CH2CH3) or permanently charged [(-)+N(CH3)3] headgroup and the distance between the headgroup and the active moiety was systematically varied with alkyl spacer chains containing 2, 6, 8, 10, 12, or 16 methylene (-CH2) groups. These compounds were previously shown, by radioligand binding experiments, to interact with the high affinity DHP binding site in intact neonatal rat heart cells. In the present experiments, access to the DHP binding site was assayed by inhibition of L-type calcium channel currents using whole-cell patch-clamp procedures in guinea pig ventricular myocytes. Intracellular application was achieved by dialysis via charged DHP-containing whole-cell patch pipettes, and cell dialysis was monitored by using a charged DHP labeled with a rhodamine fluorophore. Our results show that access of extracellularly applied charged, but not neutral, DHPs to the DHP binding domain depends markedly on the alkyl spacer chain, with the optimal length being near 10 methylene groups. Intracellular application failed to inhibit channel activity for spacer chain lengths up to 16 methylene groups. From our results, we conclude that the DHP binding domain of cardiac L-type calcium channels is not on the extracellular membrane surface but is probably within the lipid bilayer, approximately 11-14 A from the extracellular surface.

Modulation of L-type Ca2+ channels in clonal rat pituitary cells by membrane depolarization.

The modulation of L-type Ca2+ channels by membrane depolarization, in terms of channel number, function, and interaction with 1,4-dihydropyridine ligands, has been characterized in clonal rat pituitary cells (GH4C1) and rat cerebellar granule cells. Membrane depolarization by 50 mM extracellular K+ for 120 min caused an approximately 90% reduction in the total number of [3H]PN200-110 binding sites (Bmax) and an approximately 20-fold increase in binding affinity in a whole-cell binding assay. Similar results were obtained in a primary culture of rat cerebellar granule cells. In GH4C1 cells the dissociation constant (Kd) and Bmax were changed from 2.15 nM and 214 fmol/mg at 5 mM K+ to 110 pM and 24 fmol/mg at 50 mM K+, respectively. The changes in affinity and Bmax were both dependent on the extracellular K+ concentration. The affinity change resulted from an increased association rate constant (increased from 0.17 to 3.11 x 10(8) M-1 min-1 after depolarization) and an unchanged dissociation rate constant (0.032 min-1). Depolarization for 2 hr reduced the number of [3H]PN200-110 binding sites in the membrane fraction by approximately 50%, but no significant change was detected in total cell homogenates, suggesting removal of L-type Ca2+ channels from the cell surface after depolarization. Blockade of the internalization process by concanavalin A and phenylarsine oxide inhibited the depolarization-induced reduction of L-type Ca2+ channels on the cell surface. A decrease in the number of functional channels on the cell surface, as revealed by stimulated 45Ca2+ uptake, accompanied the change in [3H]PN200-110 binding. Reduction of 45Ca2+ uptake had two exponential components, i.e., rapid (with a time constant of about 2.5 min), with a rapid rate of recovery, and slow (with a time constant of 54 min), with a correspondingly slow rate of recovery. Depolarization of the cells with veratridine (50 microM) or treatment of the cells with the Ca2+ ionophore A23187 (10 microM) had effects similar to those of K+ depolarization on [3H]PN200-110 binding sites and stimulated 45Ca2+ uptake. The change in [3H]PN200-110 binding sites in whole-cell and membrane preparations occurred rapidly, becoming prominent within 45 min, and largely recovered when the cells were repolarized. The down-regulation of L-type Ca2+ channels is dependent on Ca2+ entry via a calmodulin-dependent process.

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.

Regulation of K+ and Ca2+ channels in experimental cardiac failure.

To examine the status of ATP-sensitive K+ (K+ATP) channels and 1,4-dihydropyridine-sensitive Ca2+ (Ca2+DHP) channels during experimental cardiac failure, we have measured the radioligand binding properties of [3H]glyburide and [3H]PN 200 110, respectively, in tissue homogenates from the rat cardiac left ventricle, right ventricle, and brain 4 wk after myocardial infarction induced by left coronary artery ligation. The maximal values (Bmax) for [3H]glyburide and [3H]PN 200 110 binding were reduced by 39 and 40%, respectively, in the left ventricle, and these reductions showed a good correlation with the right ventricle-to-body weight ratio in heart-failure rats. The ligand binding affinities were not altered. In the hypertrophied right ventricle, Bmax values for both the ligands were not significantly different when data were normalized to DNA content or right ventricle weights but showed an apparent reduction when normalized to unit protein or tissue weight. Moderate reductions in channel densities were observed also in whole brain homogenates from heart failure rats. Assessment of muscarinic receptors, beta-adrenoceptors and alpha 1-adrenoceptors by [3H]quinuclidinyl benzilate, [3H]dihydroalprenolol, and [3H]prazosin showed reductions in left ventricular muscarinic and beta-adrenoceptor densities but not in alpha 1-adrenoceptor densities, consistent with earlier observations. It is suggested that these changes may in part contribute to the pathology of cardiac failure.

The effects of chronic depolarization on L-type 1,4-dihydropyridine-sensitive, voltage-dependent Ca2+ channels in chick neural retina and rat cardiac cells.

Chick neural retina cells contain functional L-type voltage-dependent Ca2+ channels sensitive to 1,4-dihydropyridines. To investigate the effects of chronic depolarization, cells were grown in medium containing elevated K+. After 4-h to 4-day treatments with elevated K+ (12-73 mM), there was a concentration-dependent decrease in high affinity [3H]PN200-110 binding. Saturation analysis of cells treated for 4 days with 40 mM K+ showed a reduction in maximum ligand binding with no change in affinity. Control and experimental Bmax values were 70.7 +/- 6.4 and 42.2 +/- 4.5 fmol/mg protein, respectively, and control and experimental KD values were 70.2 +/- 7.4 and 68.6 +/- 7.4 x 10(-12) M. The effect of chronic depolarization was time-dependent, reversible, and without effect on cellular protein content. Reduction in 45Ca2+ uptake following chronic depolarization correlated well with the reduction in [3H]PN200-110 binding. The calcium ionophore A23187, 10(-6) M for 24 h, also decreased the binding site density. The calcium channel antagonist D600 had no effect alone on [3H]PN200-110 binding; however, D600 blocked the down-regulation of calcium channels induced by chronic depolarization. The mechanism for Ca2+ channel down-regulation may involve calcium entry, since the effect was blocked by D600 and mimicked by the calcium ionophore A23187. Chronic depolarization with either elevated K+ or veratridine, or chronic treatment with A23187 had no effect on calcium channels in rat neonatal ventricular myocytes, although these cells express functional channels of the 1,4-dihydropyridine-sensitive class.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.