Affinity of Electrochemically Deposited Sol⁻Gel Silica Films towards Catecholamine Neurotransmitters.

Dopamine, norepinephrine, and epinephrine neurotransmitters can be detected by electrochemical oxidation in conventional electrodes. However, their similar chemical structure and electrochemical behavior makes a difficult selective analysis. In the present work, glassy carbon electrodes have been modified with silica layers, which were prepared by electroassisted deposition of sol⁻gel precursors. These layers were morphologically and compositionally characterized using different techniques, such as field emission scanning electron microscopy (FESEM), TEM, FTIR, or thermogravimetric analysis⁻mass spectrometry (TG-MS). The affinity of silica for neurotransmitters was evaluated, exclusively, by means of electrochemical methods. It was demonstrated that silica adsorbs dopamine, norepinephrine, and epinephrine, showing different interaction with silica pores. The adsorption process is dominated by a hydrogen bond between silanol groups located at the silica surface and the amine groups of neurotransmitters. Because of the different interaction with neurotransmitters, electrodes modified with silica films could be used in electrochemical sensors for the selective detection of such molecules.

The use of a multichannel capillary for electrophoretic separations of mixtures of clinically important substances with contactless conductivity and UV photometric detection.

A fused-silica capillary with a common outer diameter, 360 µm, but containing seven internal channels, each 28 µm in diameter (a multichannel capillary), has been tested on electrophoretic separations of mixtures of dopamine, adrenaline, and noradrenaline, using a contactless conductivity and UV photometric detection. It has been demonstrated that the sensitivity of the detection of these neurotransmitters in multichannel capillary, in comparison with those obtained for a standard singlechannel capillary with similar cross-sectional area, is comparable to that for the contactless conductivity and is about 50% higher for the UV photometry. The sensitivity is increased without loss of the separation efficiency, in contrast to UV detection with bubble cell. Further possibilities of using a multichannel capillary are demonstrated on separations of mixtures of inorganic cations (K⁺, Ba²âº, Na⁺, Mg²âº, and Li⁺) and mixtures of glucose and ribose. The main advantage of multi-channel capillary in comparison with a singlechannel capillary with the same cross-sectional area becomes apparent in separations in background electrolytes of high conductivity.

An insight into the mechanism of CEC separation of template analogues on a norepinephrine-imprinted monolith.

A monolith molecularly imprinted polymer (MIP) column was prepared from template (-)-norepinephrine, functional monomer (itaconic acid), and a cross-linker (either ethylene glycol dimethacrylate or divinylbenzene) in porogen N,N-dimethylformamide. Understanding the molecular recognition of a template using an MIP seems feasible. However, it is hard to explain the recognition properties of their analogues on an MIP. The separation mechanism was investigated with the addition of charged surfactants, native and derivatised ß-cyclodextrin (ß-CD), achiral crown ether, etc. to determine the retention behaviour of the template analogues. The addition of organic modifiers and the adjustment of separation conditions were used to manipulate the selectivity. No chiral recognition was observed under most of the test conditions except the experiment with the charged ß-CD on the divinylbenzene-MIP column. The different experimental conditions led to differences in the mobilities of the analytes and resulted in remarkable enantiomeric separation of the template. We confirmed the presence of mixed-mode selectivity of the stationary phase based on hydrogen bonding, hydroelectric and hydrophobic interactions, and the electrophoretic mode.

Integration of on-chip peristaltic pumps and injection valves with microchip electrophoresis and electrochemical detection.

A microfluidic approach that integrates peristaltic pumping from an on-chip reservoir with injection valves, microchip electrophoresis and electrochemical detection is described. Fabrication and operation of both the peristaltic pumps and injection valves were optimized to ensure efficient pumping and discrete injections. The final device uses the peristaltic pumps to continuously direct sample from a reservoir containing a mixture of analytes to injection valves that are coupled with microchip electrophoresis and amperometric detection. The separation and direct detection of dopamine and norepinephrine were possible with this approach and the utility of the device was demonstrated by monitoring the stimulated release of these neurotransmitters from a layer of cells introduced into the microchip. It is also shown that this pumping/reservoir approach can be expanded to multiple reservoirs and pumps, where one reservoir can be addressed individually or multiple reservoirs sampled simultaneously.

Simultaneous separation of epinephrine and norepinephrine enantiomers by EKC: application to the analysis of pharmaceutical formulations.

A new analytical methodology using EKC with a CD as chiral selector was developed enabling the simultaneous enantiomeric separation of two chiral neurotransmitters (epinephrine and norepinephrine). Different experimental conditions (type and concentration of the chiral selector, nature and concentration of buffer and separation voltage) were optimized. The use of 10 mM carboxyethyl-beta-CD in 100 mM Tris-phosphate buffer (pH 5.0) with an applied voltage of 30 kV and a temperature of 15 degrees C allowed the separation of epinephrine and norepinephrine enantiomers with high chiral resolution (Rs>2.5). Moreover, after evaluating the analytical characteristics of the method, it was applied to the quantitation and purity testing of L-epinephrine as active pharmaceutical ingredient in pharmaceutical formulations where D-epinephrine and L/D-norepinephrine can be present as impurities.

Localization and characterization of carbohydrates in adrenal medullary cells.

The localization and characterization of carbohydrates in adrenal medullary cells were studied by histochemical and cytochemical methods. Adrenaline (A)-and noradrenaline (N)-storing granules were argentaphobic when ultrathin sections of Araldite-embedded medullae were stained according to the periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery. A small amount of glycogen in the form of single beta-particles as well as lysosomes were, however, visualized by this technique. The entire core of the A granules was markedly positive after ultrathin sections of glutaraldehyde-fixed, glycol methacrylate (GMA)-embedded medullae were stained with phosphotungstic acid (PTA) at low pH (0.3). The N granules, in contrast, were mostly unreactive. In the A cells, PTA stained a large part of the Golgi complex, whereas in the N cells the Golgi complex was mostly unstained. In both cell types, the cell coat, lysosomes, and multivesticular bodies reacted to PTA. The periodic acid-Schiff (PAS) technique showed A but not N granules in semithin sections of GMA- or Araldite-embedded medullae. The PTA and PAS stains were abolished by acetylation, restored by saponification, unchanged by methylation, and greatly diminished by sulfation. In ultrathin sections of GMA- or Araldite-embedded medullae incubated with colloidal iron according to various techniques, the cell coat and lysosomes of both cell types were stained, unlike all the other cytoplasmic organelles. These results indicate that A granules and the Golgi complex of A cells, unlike the same structures in N cells, are rich in glycoproteins which are probably not acidic.

Thermoregulation: effects of environmental temperature on turnover of hypothalamic norepinephrine.

The hypothesis that norepinephrine is a transmitter in the temperature regulating center of the hypothalamus is based on observations of changes in the rectal temperatures of animals after injections of norepinephrine into the hypothalamus. By introducing tritiated norepinephrine as a label into the endogenous norepinephrine stores in the brain and then measuring the disappearance of tritiated norepinephrine from discrete areas, one can monitor the activity of norepinephrine-containing neurons in those areas. In the rat exposed to heat, the turnover of endogenous norepinephrine appears to be increased selectively in the hypothalamus, whereas exposure to cold has no effect.

Polymethacryloyl-L-Phenylalanine [PMAPA]-Based Monolithic Column for Capillary Electrochromatography.

The ability to detect catecholamines (CAs) and their metabolites is vital to understand the mechanism behind the neuronal diseases. Neurochemistry aims to provide an improved pharmacological, molecular and physiological understanding of complex brain chemistries by analytical techniques. Capillary electrophoresis (CE) is one such analytical technique that enables the study of various chemical species ranging from amino acids and peptides to natural products and drugs. CE can easily adapt the changes in research focus and in recent years remains an applicable technique for investigating neuroscience and single cell neurobiology. The prepared phenylalanine-based hydrophobic monolithic column, Polymethacryloyl-L-phenylalanine [PMAPA], was used as a stationary phase in capillary electrochromatography to separate CAs that are similar in size and shape to each other including dopamine (DA) and norepinephrine (NE) via hydrophobic interactions. Separation carried out in a short period of 17 min was performed with the electrophoretic mobility of 5.54 × 10-6 m2 V-1 s-1 and 7.60 × 10-6 m2 V-1 s-1 for DA and NE, respectively, at pH 7.0, 65% acetonitrile ratio with 100 mbar applied pressure by the developed hydrophobic monolithic column without needing any extra process such as imprinting or spacer arms to immobilize ligands used in separation.

Nicotinamide adenine dinucleotide immobilized tungsten trioxide nanoparticles for simultaneous sensing of norepinephrine, melatonin and nicotine.

Herein, we report the anionic surfactant, ethylene diamine tetraacetic acid (EDTA), mediated synthesis of WO3 nanoparticles and its subsequent modification through gamma irradiation (GI) and electrochemical immobilization with nicotinamide adenine dinucleotide (NAD). Glassy carbon electrode (GCE) modified with GI-WO3 NPs and the enzyme NAD exhibited strong electro-oxidation of three important biomolecules such as norepinephrine (NEP), melatonin (MEL) and nicotine (NIC) in 0.1 M phosphate buffer saline (PBS) at physiological pH of 7. Square wave voltammetry (SWV) studies exhibited three well-defined peaks at potentials of 120, 570 and 840 mV, corresponding to the oxidation of NEP, MEL and NIC respectively, indicating that simultaneous determination of these compounds is feasible at the NAD/GI EDTA-WO3/GCE. The proposed sensor displayed a wide linear range of 0.010-1000 µM with the lowest detection limit of 1.4 nM for NEP, 2.6 nM for MEL and 1.7 nM for NIC respectively. Furthermore, the modified electrode was successfully applied to detect NEP, MEL and NIC in pharmaceutical and cigarette samples with excellent selectivity and reproducibility.

A signal amplification system constructed by bi-enzymes and bi-nanospheres for sensitive detection of norepinephrine and miRNA.

Achieving the enhanced sensitivity and stability is always the pursuit for the fabrication of enzymatic biosensors. However, their sensitivity was still restricted by the fluctuant detection target (e.g. concentration), complex detection environment and limited recognition capability of enzymes. Herein, an effective and facile approach was designed to construct a bi-enzymatic and bi-nanospherical signal amplification system for fabrication of biosensors based on the designed polydopamine(PDA)-laccase@Au-glucose dehydrogenase. Therein, laccase-catalytic polymerized PDA nanoparticles (NPs) provided the supporting matrix for immobilization of laccase and AuNPs. The AuNPs with good conductivity and large surface area were used not only as a platform for enhanced loading capacity of glucose dehydrogenase but also as a conducting medium for electron transfer acceleration between enzymes and electrode. Moreover, the coordinated catalysis of bi-enzymes (laccase and glucose dehydrogenase) could avoid the fluctuated concentration of detection target (e.g. norepinephrine), while the application of bi-nanospheres loaded with large amount of enzymes could effectively amplify the signal of biosensors. Taking advantages of these merits, the as-prepared biosensors showed preeminent reproducibility, larger detection range from 0.5 nM to 0.5 µM, and lower detection limit of 0.07 nM (S/N = 3) for the norepinephrine detection. Besides, the constructed PDA-laccase@Au-glucose dehydrogenase was also successfully applied as the sensing probes for the detection of microRNA (miRNA), especially for single-nucleotide mismatched miRNA via specific recognition.

Transcriptome-wide association study of attention deficit hyperactivity disorder identifies associated genes and phenotypes.

Attention deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental psychiatric disorder. Genome-wide association studies (GWAS) have identified several loci associated with ADHD. However, understanding the biological relevance of these genetic loci has proven to be difficult. Here, we conduct an ADHD transcriptome-wide association study (TWAS) consisting of 19,099 cases and 34,194 controls and identify 9 transcriptome-wide significant hits, of which 6 genes were not implicated in the original GWAS. We demonstrate that two of the previous GWAS hits can be largely explained by expression regulation. Probabilistic causal fine-mapping of TWAS signals prioritizes KAT2B with a posterior probability of 0.467 in the dorsolateral prefrontal cortex and TMEM161B with a posterior probability of 0.838 in the amygdala. Furthermore, pathway enrichment identifies dopaminergic and norepinephrine pathways, which are highly relevant for ADHD. Overall, our findings highlight the power of TWAS to identify and prioritize putatively causal genes.

Norepinephrine alkaloids as antiplasmodial agents: Synthesis of syncarpamide and insight into the structure-activity relationships of its analogues as antiplasmodial agents.

Syncarpamide 1, a norepinephrine alkaloid isolated from the leaves of Zanthoxylum syncarpum (Rutaceae) exhibited promising antiplasmodial activities against Plasmodium falciparum with reported IC50 values of 2.04 µM (D6 clone), 3.06 µM (W2 clone) and observed by us 3.90 µM (3D7 clone) and 2.56 µM (K1 clone). In continuation of our work on naturally occurring antimalarial compounds, synthesis of syncarpamide 1 and its enantiomer, (R)-2 using Sharpless asymmetric dihydroxylation as a key step has been accomplished. In order to study structure-activity-relationship (SAR) in detail, a library of 55 compounds (3-57), which are analogues/homologues of syncarpamide 1 were synthesized by varying the substituents on the aromatic ring, by changing the stereocentre at the C-7 and/or by varying the acid groups in the ester and/or amide side chain based on the natural product lead molecule and further assayed in vitro against 3D7 and K1 strains of P. falciparum to evaluate their antiplasmodial activities. In order to study the effect of position of functional groups on antiplasmodial activity profile, a regioisomer (S)-58 of syncarpamide 1 was synthesized however, it turned out to be inactive against both the strains. Two compounds, (S)-41 and its enantiomer, (R)-42 having 3,4,5-trimethoxy cinnamoyl groups as side chains showed better antiplasmodial activity with IC50 values of 3.16, 2.28 µM (3D7) and 1.78, 2.07 µM (K1), respectively than the natural product, syncarpamide 1. Three compounds (S)-13, (S)-17, (S)-21 exhibited antiplasmodial activities with IC50 values of 6.39, 6.82, 6.41 µM against 3D7 strain, 4.27, 7.26, 2.71 µM against K1 strain and with CC50 values of 147.72, 153.0, >200 µM respectively. The in vitro antiplasmodial activity data of synthesized library suggests that the electron density and possibility of resonance in both the ester and amide side chains increases the antiplasmodial activity as compared to the parent natural product 1. The natural product syncarpamide 1 and four analogues/homologues out of the synthesized library of 55, (S)-41, (R)-42, (S)-55 and (S)-57 were assayed in vivo assay against chloroquine-resistant P. yoelii (N-67) strain of Plasmodium. However, none of the five molecules, 1, (S)-41, (R)-42, (S)-55 and (S)-57 exhibited any promising in vivo antimalarial activity against P. yoelii (N-67) strain. Compounds 4, 6, 7 and 11 showed high cytotoxicities with CC50 values of 5.87, 5.08, 6.44 and 14.04 µM, respectively. Compound 6 was found to be the most cytotoxic as compared to the standard drug, podophyllotoxin whereas compounds 4 and 7 showed comparable cytotoxicities to podophyllotoxin.

Biochemical studies on the release of catecholamines from the rat carotid body in vitro.

The effects of hypoxia and carbachol on the release of newly synthesized catecholamines from superfused rat carotid bodies have been examined. Hypoxic superfusion medium was found to evoke catecholamine release which was dependent on the extracellular calcium concentration and was reduced by nitrendipine and atropine. Superfusion with the muscarinic agonist, carbachol, stimulated catecholamine release independently of the oxygen tension of the medium. The effect of carbachol on catecholamine release was abolished by atropine, suggesting that it was mediated by activation of cholinergic receptors of the muscarinic type. Both hypoxia and carbachol stimulated the release of 45Ca from carotid bodies prelabelled with 45Ca. The release of 45Ca with either stimulus was reduced by atropine and nitrendipine. These results suggest that although extracellular calcium plays an important role in the exocytotic secretory process of the carotid body, the mobilization of intracellular calcium pools may also contribute to the secretory response.

Separation and determination of norepinephrine, epinephrine and isoprinaline enantiomers by capillary electrophoresis in pharmaceutical formulation and human serum.

A capillary electrophoresis method with ultraviolet (UV) detection was developed and optimized for the enantiomer separation of norepinephrine (NE), epinephrine (EP) and isoprenaline (IP) using dual cyclodextrins (CDs) of 2-hydroxypropyl-beta-CD (HP-beta-CD) and heptakis (2,6-di-o-methyl)-beta-CD (DM-beta-CD) as chiral selectors. Optimal separation was obtained using a running buffer of 50mM phosphate containing 30mM HP-beta-CD and 5mM DM-beta-CD at pH 2.90 and a field strength of 20kV in 45cmx75mum (40cm effective length) uncoated capillary. The UV absorbance detection was set at 205nm. A 0.1% (w/w) polyethylene glycol or 0.1% (v/v) acetonitrile was used to enhance the detection sensitivity. There was a wide and excellent linear calibration graph for each enantiomer in the range 1.0x10(-3) to 1.0x10(-6)M and the detection limit (S/N=3) was found from 8.5x10(-7) to 9.5x10(-7)M. The method has been applied for the determination of isoprenaline in isoprenaline hydrochloride aerosol and to the analysis of serum samples. The recoveries of NE and EP in serum and IP in drug were ranged from 90 to 110%. The relative standard deviations of all the analyte peaks were less than 2.8% for migration time and less than 4.8% for peak area.

A sensitive radioenzymatic assay for dopamine, norepinephrine, and epinephrine in plasma and tissue.

A double-isotope, radioenzymatic assay for measuring dopamine, norepinephrine, and epinephrine in one sample is described. The assay procedure includes incubation, solvent extraction, and thin-layer chromatography. Dopamine, norepinephrine, and epinephrine were incubated with catechol-O-methyl transferase (COMT) and [3H]S-acenosyl methionine ([3H]SAM) and were converted to the O-methylated tritiated derivatives: [3H]methoxytyramine, [3H]normetanephrine, and [3H]metanephrine, respectively. After several extraction steps the O-methylated products were purified by means of two-dimensional, thin-layer chromatography using silica gel. The thin-layer chromatographic system resulted complete separation of the three O-methylated compounds with an overlap of only 1-2%. The assay was linear from 0 to 5 ng for each catecholamine and had a sensitivity of 10-30 pg. The addition of large amounts of plasma reduced the activity of COMT, but increasing the magnesium concentration in the incubation mixture and the addition of EGTA to plasma samples improved the recoveries. Each sample was corrected for losses incurred during extraction and chromatography by using [14C]methoxytyramine, [14C]normetanephrine, and [14Ci1metanephrine that were added at the end of incubation. Several catechol compounds known to be O-methylated by COMT were examined for crossreactivity. Of the substances tested, only dihydroxyphenylalanine (DOPA) exhibited cross-reactivity. However, the apparent 30% cross-reactivity of DOPA with dopamine was due to the presence of decarboxylase activity in the COMT preparation. As little as 50 mul of trunk plasma from decapitated rats was sufficient for the determination of the three catecholamines.

The influence of atherogenic diet and "essential" phospholipids upon the contents of noradrenaline and dopamine in the brain of rats and their exploratory activity.

The influence of atherogenic diet and "essential" phospholipids (EPL) both on exploratory activity and catecholamine content in the brain of rats has been examined. The atherogenic diet caused a decrease of the animals' exploratory activity as well as a diminution of catecholamines. It was observed that these changes correlated. Prophylactic administration of EPL is capable of preventing such changes. Therapeutic administration of EPL increased exploratory activity and catecholamines in the atherogenic group which subsequently received the basic laboratory diet.

The development of enzyme-linked immunosorbent assays (ELISA) for the catecholamines adrenalin and noradrenalin.

Monoclonal antibodies have been raised against adrenalin and noradrenalin and used as the basis of enzyme-linked immunosorbent assays (ELISA) to detect and estimate the concentrations of these catecholamines. Inhibition assays are described, with sensitive quantification in the range from 1 mg/ml to 100 pg/ml. Cross-reactivity assays reveal that neither assay is subject to interference by catecholamine metabolites at concentrations less than 100 ng/ml and 1 micrograms/ml respectively. Isolation and quantification of both catecholamines from clinical samples is discussed and the potential application of these ELISAs in a clinical setting is proposed.

Sympathetic innervation of the amphibian spleen: developmental studies in Xenopus laevis.

Spleens from larval and adult South African clawed frogs (Xenopus laevis) were examined using sucrose-potassium phosphate-glyoxylic acid (SPG) histofluorescence for norepinephrine. Innervation of the larval Xenopus spleen is barely detectable at stage 54 and gradually increases during prometamorphosis (stage 57/58) until metamorphic climax (stage 66). This development of innervation late in the larval life of the animal was highly sensitive to environmental conditions and to rapidity at which development occurred. Prevention of overt metamorphosis by sodium perchlorate blockade prevented the development of noradrenergic (NA) splenic innervation in some, but not all, tadpoles examined. Depletion of T-lymphocytes by early larval thymectomy did not alter the kinetics or pattern of splenic NA innervation.

Increased norepinephrine content in diabetic rat heart.

In an attempt to clarify the mechanism of cardiac neuropathy in diabetes mellitus, Wister male rats were made diabetic by streptozotocin injection for 11 to 13 weeks, and catecholamine concentrations of hearts were determined by high performance liquid chromatography. No apparent histological changes were found in hearts and kidneys of any group of rats. Controls used were age-matched normal rats and Goldblatt-hypertensive rats, because streptozotocin induced diabetic rats appeared to be significantly hypertensive. Heart norepinephrine concentrations of diabetic rats and diabetic-Goldblatt-hypertensive rats were markedly higher (8,380 +/- 300 pmol/g tissue and 6,980 +/- 390, respectively) compared with those of controls and Goldblatt-hypertensive rats (2,700 +/- 470 and 2,010 +/- 300, respectively). These results suggest some disturbances in catecholamine secretion in diabetic hearts before typical microangiopathic changes take place.

The separation of epinephrine from norepinephrine and dopamine from DOPA on Sephadex G-10.

Epinephrine and norepinephrine were separated by acid elution through a Sephadex G-10 column with high recovery (better than 90%), excellent reproducibility, and little overlap (less than 10%). Once packed, the columns could be re-used indefinitely. Total elution time was about six hours and the columns could be left untended since the gravity flow stops automatically once the level of the eluant reaches the gel bed. The resulting dilution was five-fold. 3,4-Dihydroxyphenylalanine (DOPA) was completely separated from 3,4-dihydroxyphenylethylamine (dopamine) by the same procedure.