Nitrogen-doped carbon dots aid in the separation of ssDNA molecules of different length by capillary transient isotachophoresis (ctITP) with laser-induced fluorescence (LIF) detection.

This study demonstrates a novel application of nitrogen-doped carbon dots (NCDs) to enable the separation of different lengths of single-stranded DNA (ssDNA) by eletrokinetic means. Carbon dots have recently found widespread application in the fields of sensing, diagnostics, and healthcare due to their biocompatibility and low toxicity. In light of growing interest in the use of ssDNA aptamers over antibodies in the fields of biosensor development and drug delivery, it is important to establish a simple and effective method for aptamer separation. In this study, we employed NCDs as buffer additives in a capillary electrophoresis (CE)-based method, giving rise to the separation of FAM-labeled ssDNA samples ranging from 32 to 100 bases in length, with resolutions ranging from 1.30 - 1.77. In particular, we adopted a capillary transient isotachophoresis (ctITP) system with laser-induced fluorescence (LIF) detection, with both the separation and sample buffers modified by the addition of 30 µg/mL NCDs. These nanomaterials were prepared by a simple hydrothermal method from a mixture of citric acid and ethylenediamine. The NCDs themselves are highly fluorescent and photostable. As components in the background electrolyte, they did not interfere with the fluorescence emission of the FAM-labeled DNA samples. Under the conditions employed, no separation could be achieved in the absence of the NCDs nor with undoped CDs. The results show that NCDs function as buffer additives capable of enhancing electrokinetic-based separations of ssDNA, and hence, provide a new application for these carbon nanomaterials.

Two-Dimensional Polyacrylamide Gel Electrophoresis for Metalloprotein Analysis Based on Differential Chemical Structure Recognition by CBB Dye.

In an effort to develop an analytical method capable of finding new metalloproteins, this is the first report of a new diagonal gel electrophoresis method to isolate and identify metalloproteins, based on the molecular recognition of holo- and apo-metalloproteins (metalbound and -free forms, respectively) by CBB G-250 dye and employing metal ion contaminant sweeping-blue native-polyacrylamide gel electrophoresis (MICS-BN-PAGE). The difference in electrophoretic mobilities between holo- and apo-forms was exaggerated as a result of interactions between the metalloproteins and the dye with no metal ion dissociation. The different binding modes of proteins with CBB G-250 dye, primarily related to hydrogen bonding, were confirmed by capillary zone electrophoresis (CZE) and molecular docking simulations. Due to in-gel holo/apo conversion between the first and second dimensions of PAGE, holo-metalloproteins in the original sample were completely isolated as spots off the diagonal line in the second dimension of PAGE. To prove the high efficiency of this method for metalloprotein analysis, we successfully identified a copper-binding protein from a total bacterial soluble extract for the first time.

Carbon Dot-Mediated Capillary Electrophoresis Separations of Metallated and Demetallated Forms of Transferrin Protein.

Carbon dots (CDs) are fluorescent nanomaterials used extensively in bioimaging, biosensing and biomedicine. This is due in large part to their biocompatibility, photostability, lower toxicity, and lower cost, compared to inorganic quantum dots or organic dyes. However, little is known about the utility of CDs as separation adjuvants in capillary electrophoresis (CE) separations. CDs were synthesized in-house according to a 'bottom-up' method from citric acid or other simple carbon precursors. To demonstrate the applicability of CDs as separation adjuvants, mixtures of holo- (metallated) and apo- (demetallated) forms of transferrin (Tf, an iron transport protein) were analyzed. In the absence of CDs, the proteins were not resolved by a simple CE method; however, upon addition of CDs to the separation buffer, multiple forms of Tf were resolved indicating that CDs are valuable tools to facilitate the separation of analytes by CE. CE parameters including sample preparation, buffer identity, ionic strength, pH, capillary inside diameter, and temperature were optimized. The results suggest that dots synthesized from citric acid provide the best resolution of various different forms of Tf and that CDs are versatile and promising tools to improve current electrophoretic separation methods, especially for metalloprotein analysis.

Development of High-Throughput Method for Measurement of Vascular Nitric Oxide Generation in Microplate Reader.

BACKGROUND: Despite the importance of nitric oxide (NO) in vascular physiology and pathology, a high-throughput method for the quantification of its vascular generation is lacking. OBJECTIVE: By using the fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM), we have optimized a simple method for the determination of the generation of endothelial nitric oxide in a microplate format. METHODS: A nitric oxide donor was used (3-morpholinosydnonimine hydrochloride, SIN-1). Different factors affecting the method were studied, such as the effects of dye concentration, different buffers, time of reaction, gain, and number of flashes. RESULTS: Beer's law was linear over a nanomolar range (1-10 nM) of SIN-1 with wavelengths of maximum excitation and emission at 495 and 525 nm; the limit of detection reached 0.897 nM. Under the optimized conditions, the generation of rat aortic endothelial NO was measured by incubating DAF-FM with serial concentrations (10-1000 µM) of acetylcholine (ACh) for 3 min. To confirm specificity, Nω-Nitro-l-arginine methyl ester (l-NAME)-the standard inhibitor of endothelial NO synthase-was found to inhibit the ACh-stimulated generation of NO. In addition, vessels pre-exposed for 1 h to 400 µM of the endothelial damaging agent methyl glyoxal showed inhibited NO generation when compared to the control stimulated by ACh. CONCLUSIONS: The capability of the method to measure micro-volume samples makes it convenient for the simultaneous handling of a very large number of samples. Additionally, it allows samples to be run simultaneously with their replicates to ensure identical experimental conditions, thus minimizing the effect of biological variability.

Selection of a Novel Aptamer Against Vitronectin Using Capillary Electrophoresis and Next Generation Sequencing.

Breast cancer (BC) results in ~40,000 deaths each year in the United States and even among survivors treatment of the disease may have devastating consequences, including increased risk for heart disease and cognitive impairment resulting from the toxic effects of chemotherapy. Aptamer-mediated drug delivery can contribute to improved treatment outcomes through the selective delivery of chemotherapy to BC cells, provided suitable cancer-specific antigens can be identified. We report here the use of capillary electrophoresis in conjunction with next generation sequencing to develop the first vitronectin (VN) binding aptamer (VBA-01; Kd 405 nmol/l, the first aptamer to vitronectin (VN; Kd = 405 nmol/l) , a protein that plays an important role in wound healing and that is present at elevated levels in BC tissue and in the blood of BC patients relative to the corresponding nonmalignant tissues. We used VBA-01 to develop DVBA-01, a dimeric aptamer complex, and conjugated doxorubicin (Dox) to DVBA-01 (7:1 ratio) using pH-sensitive, covalent linkages. Dox conjugation enhanced the thermal stability of the complex (60.2 versus 46.5°C) and did not decrease affinity for the VN target. The resulting DVBA-01-Dox complex displayed increased cytotoxicity to MDA-MB-231 BC cells that were cultured on plasticware coated with VN (1.8 × 10-6mol/l) relative to uncoated plates (2.4 × 10-6 mol/l), or plates coated with the related protein fibronectin (2.1 × 10-6 mol/l). The VBA-01 aptamer was evaluated for binding to human BC tissue using immunohistochemistry and displayed tissue specific binding and apparent association with BC cells. In contrast, a monoclonal antibody that preferentially binds to multimeric VN primarily stained extracellular matrix and vessel walls of BC tissue. Our results indicate a strong potential for using VN-targeting aptamers to improve drug delivery to treat BC.

Using capillary electrophoresis to characterize polymeric particles.

Capillary electrophoresis (CE) was used for the characterization of a variety of polymeric micron and sub-micron particles based on size, surface functionality, and binding properties. First, a robust capillary zone electrophoresis (CZE) method was developed for the baseline separation and quantitation of commercially available polystyrene particles with various surface modifications (including amino, carboxylate, and sulfate functional groups) and various sizes (0.2, 0.5, 1.0, and 3.0µm). The separation of DNA-templated polyacrylamide particles from untemplated particles (as used for the Ion Torrent Personal Genome Machine) was demonstrated. Finally, using the 29-base thrombin aptamer and thrombin protein as a model system, a study was undertaken to determine dissociation constants for the aptamer and protein in free solution and when the aptamer was conjugated to a particle, with the goal of better understanding how the use of solid substrates, like particles, affects selection and binding processes. Dissociation constants were determined and were found to be approximately 5-fold higher for the aptamer conjugated to a particle relative to that in free solution.

Molecular design of boronic acid-functionalized squarylium cyanine dyes for multiple discriminant analysis of sialic acid in biological samples: selectivity toward monosaccharides controlled by different alkyl side chain lengths.

We designed a new series of boronic acid-functionalized squarylium cyanine dyes (SQ-BA) with different lengths of alkyl chain residues, suitable for multiple discriminant analysis (MDA) of sialic acid (Neu5Ac) in biological samples. The SQ-BA dyes form aggregates based on hydrophobic interactions, which result in quenched fluorescence in aqueous solutions. When the boronic acid binds with saccharides, the fluorescence intensity increases as a result of dissociation to the emissive monomeric complex. We inferred that different dye aggregate structures (H-aggregates and J-aggregates) were induced depending on the alkyl chain length, so that monosaccharides would be recognized in different ways (especially, multipoint interaction with J-aggregates). A distinctive emission enhancement of SQ-BA dyes with shorter-alkyl-chains in the presence of Neu5Ac was observed (2.4-fold fluorescence enhancement; with formation constant 10(1.7) M(-1)), with no such enhancement for SQ-BA dyes with longer-alkyl-chain. In addition, various enhancement factors for other monosaccharides were observed depending on the alkyl chain length. Detailed thermodynamic and NMR studies of the SQ-BA complexes revealed the unique recognition mechanism: the dye aggregate with a shorter-alkyl-chain causes the slipped parallel structure and forms a stable 2:1 complex with Neu5Ac, as distinct from longer-alkyl-chain dyes, which form a 1:1 monomeric complex. MDA using the four SQ-BA dyes was performed for human urine samples, resulting in the successful discrimination between normal and abnormal Neu5Ac levels characteristic of disease. Thus, we successfully controlled various responses to similar monosaccharides with a novel approach that chemically modified not the boronic acid moiety itself but the length of the alkyl chain residue attached to the dye in order to generate specificity.

Capillary electrophoresis-mass spectrometry determination of morphine and its isobaric glucuronide metabolites.

The determination of morphine and its isobaric metabolites morphine-3-beta-d-glucuronide (M3G) and morphine-6-beta-d-glucuronide (M6G) is useful for therapeutic drug monitoring and forensic identification of drug use. In particular, capillary electrophoresis with mass spectrometry (CE-MS) represents an attractive tool for opioid analysis. Whereas volatile background electrolytes in CE often improve electrospray ionization for coupled MS detection, such electrolytes may reduce CE separation efficiency and resolution. To better understand the effects of background electrolyte (BGE) composition on separation efficiency and detection sensitivity, this work compares and contrasts method development for both volatile (ammonium formate and acetate) and nonvolatile (ammonium phosphate and borate) buffers. Peak efficiencies and migration times for morphine and morphine metabolites were optimal with a 25mM ammonium borate buffer (pH=9.5) although greater sensitivities were achieved in the ammonium formate buffer. Optimized CE methods allowed for the resolution of the isobaric morphine metabolites prior to high mass accuracy, electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS detection applicable to the analysis of urine samples in under seven minutes. Urine sample preparation required only a 10-fold dilution with BGE prior to analysis. Limits of detection (LOD) in normal human urine were found to be 1.0µg/mL for morphine and 2.5µg/mL for each of M3G and M6G by CE-ESI-QTOF-MS. These LODs were comparable to those for CE-UV analysis of opioid standards in buffer, whereas CE-ESI-QTOF-MS analysis of opioid standards in buffer yielded LODs an order of magnitude lower. Patient urine samples (N=12) were analyzed by this new CE-ESI-QTOF-MS method and no significant difference in total morphine content relative to prior liquid chromatography-mass spectrometry (LC-MS) results was found as per a paired-t test at the 99% confidence level. Whereas the LC-MS method applied to these samples determined only total morphine content, this new CE-ESI-QTOF-MS method allowed for species differentiation in addition to total morphine determination. By this method, it was found that M3G and M6G metabolites were present in a 5:1 concentration ratio, on average, in patient samples. Therefore, the CE-ESI-QTOF-MS method not only allows for total morphine concentration determination comparable to established LC-MS methods, but also allows for differentiation between morphine and its trace glucuronides, yielding additional biochemical information about drug metabolism.

Combining capillary electrophoresis and next-generation sequencing for aptamer selection.

Next-generation sequencing (NGS) machines can sequence millions of DNA strands in a single run, such as oligonucleotide (oligo) libraries comprising millions to trillions of discrete oligo sequences. Capillary electrophoresis is an attractive technique to select tight binding oligos or "aptamers" because it requires minimal sample volumes (e.g., 100 nL) and offers a solution-phase selection environment through which enrichment of target-binding oligos can be determined quantitatively. We describe here experiments using capillary transient isotachophoresis (ctITP)-based nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) as a method for selecting aptamers from a randomized library containing a known (29mer) thrombin-binding aptamer. Our capillary electrophoresis (CE)-selected samples were sequenced by the Ion Torrent Personal Genome Machine (PGM) and analyzed for selection efficiency. We show that a single round of CE selection can enrich a randomer synthetic DNA oligo mixture for thrombin-binding activity from 0.4% aptamer content before selection to >15% aptamer content.

Facilitating aptamer selection and collection by capillary transient isotachophoresis with laser-induced fluorescence detection.

An efficient separation method that utilizes capillary transient isotachophoresis (ctITP) was developed for the preselection of binding ligands. With the ultimate goal of providing enriched fractions from vast libraries for drug discovery, the preselection process described herein entails three distinct elements, which have been validated using a model thrombin protein (target) and thrombin aptamer (ligand) system. First, a high fidelity, on-column labeling scheme employing the noncovalent, fluorescent reagent SYBR Gold was demonstrated for single-stranded DNA with an 11-fold greater sensitivity than pre-column labeling procedures. Second, this on-column labeling was incorporated into a new ctITP method with laser-induced fluorescence (LIF) detection, which provided greatly enhanced resolution of protein-aptamer complex and free aptamer (in comparison to traditional capillary zone electrophoresis (CZE) methods). Third, this enhanced resolution permitted the subsequent accumulation of bound aptamer fractions via an automated collection method, with the establishment of quantitative measures of DNA accumulation. Preselected aptamer or ligand samples such as these can serve as inputs for subsequent lab-on-bead or next-generation-sequencing technologies, enabling accelerated drug discovery.

Utility of 4-chloro-7-nitrobenzofurazan (NBD-CI) for the Spectrophotometric and spectrofluorometric determination of several antihistamine and antihypertensive drugs.

New, sensitive, and selective spectrophotometric and spectrofluorometric methods have been developed for determination of clemastine hydrogen fumarate (Clem), loratadine (Lor), losartan potassium (Los), and ramipril (Ram) in both pure form and pharmaceutical formulations using 4-chloro-7-nitrobenzofurazan (NBD-CI), which is a highly sensitive chromogenic and fluorogenic reagent. The relation between absorbance at 470, 467, 471, and 469 nm and the concentration was linear over the ranges 5-35, 10-100, 10-90, and 10-120 microg/mL for Clem, Lor, Los, and Ram, respectively. The complexation products were also measured spectrofluorometrically at the emission wavelength 535 nm for Clem, Lor, and Ram and at 538 nm for Los with excitation at 477 and 452 nm for Clem and Lor, respectively, and 460 nm for both Los and Ram. The fluorescence intensity was directly proportional to the drug concentration over the ranges 0.05-0.5, 5-20, 1-6, and 2-15 microg/mL for Clem, Lor, Los, and Ram, respectively. The methods were successfully applied for the determination of the studied drugs in pharmaceutical dosage forms with excellent recovery.

An application of polymer-enhanced capillary transient isotachophoresis with an emissive boronic acid functionalized squarylium dye as an on-capillary labeling agent for gram-positive bacteria.

In this paper, the characterization and application of the "PectI" (polymer-enhanced capillary transient isotachophoresis) technique for the separation and detection of same genus, gram-positive bacteria, Bacillus globigii (Bg) and Bacillus subtilis, is demonstrated by employing a boronic acid-functionalized squarylium dye (SQ-BA) as an on-capillary labeling agent, including the quantitative performance and applicability to crude samples. The effect of borate in the separation buffer was also investigated, which revealed that borate strongly affects the separation behavior of bacteria.

A long-wavelength fluorescent squarylium cyanine dye possessing boronic acid for sensing monosaccharides and glycoproteins with high enhancement in aqueous solution.

Fluorescence sensing of saccharides and glycoproteins using a boronic acid functionalized squarylium cyanine dye ("SQ-BA") is characterized in terms of synthetic, fluorometric, thermodynamic and kinetic parameters. In our previous work, this newly synthesized dye was successfully applied to the separation and quantification of Gram-positive bacteria by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF); however, the fundamental properties of the dye and its saccharide complexes still required elucidation, as presented in this paper. The dye itself forms nonemissive, soluble aggregates in aqueous solution. With the addition of a monosaccharide, the dye aggregate dissociates to form an emissive monomer accompanied by the formation of a cyclic cis-diol ester with long-wavelength emission (λ(ex) = 630 nm, λ(em) = 660 nm). A very large fluorescence enhancement factor of 18× was observed for the sensing dye as a fructose complex at pH 10, yielding a limit of detection of 10 µM fructose. The relative order of fluorescence enhancement of SQ-BA with other monosaccharides was found to be: fructose > ribose > arabinose ≈ galactose > xylose > mannose > rhamnose > fucose ≈ glucose; and apparent affinity constants of 10(2.80), 10(2.08) and 10(0.86) M(-1) were determined for fructose, ribose and glucose, respectively. Formation of the emissive complexes occurred within minutes, proving the kinetics of the sugar-dye interactions to be suitable for on-column labeling methods in CE-LIF. Furthermore, the sensing dye was successfully applied to glycoproteins, mucin type I-S and type III, which were detected with high sensitivity in batch aqueous solution as a result of the sugar-selective boronic acid-diol esterification as well as hydrophobic interactions.

CE-ESI-MS separation of divalent organic and inorganic anions using a tricationic complexing reagent.

A tricationic ion-pairing reagent, 1,3,5-1-butyl-3-methyl-1H-imidazol-3-ium-2,4,6-trimethylbenzene, was used to form complexes with doubly charged anions for their subsequent analysis by capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) in the positive-ion mode. This methodology offers the advantages of greater versatility and sensitivity relative to direct detection of the anions in negative-ion mode, and it can be realized by a number of possible complexation strategies, including pre-column, on-column, and post-column modes. Three model anions, sulfate [SO4²â»], thiosulfate [TSFA, S2O3²â»], and benzenedisulfonate [BZDS, C6H4(SO3)2²â»], were amenable to complexation with the tricationic reagent, yielding singly charged cations with greater mass-to-charge (m/z) ratios than the native analytes. By utilizing optimized parameters obtained through previous work with dicationic reagents and singly charged anions, including the CE separation buffer composition and pH, the concentration of the dicationic reagent, the mode of complexation, the nebulizing gas pressure, and the sheath liquid composition, it was possible to develop a robust CE-ESI-MS method appropriate for the analysis of divalent anions in a mixture.

On-column labeling of gram-positive bacteria with a boronic acid functionalized squarylium cyanine dye for analysis by polymer-enhanced capillary transient isotachophoresis.

A new asymmetric, squarylium cyanine dye functionalized by boronic acid ("SQ-BA") was designed and synthesized for on-capillary labeling of gram-positive bacteria to provide for high sensitivity detection by way of a modified form of capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The CE-based separation employed a polymer-enhanced buffer with capillary transient isotachophoresis in a new hybrid method dubbed "PectI." It was found that the addition of various monosaccharides to SQ-BA in a batch aqueous solution greatly enhanced the emission of the boronic acid functionalized dye by a factor of up to 18.3 at a long wavelength (λ(ex) = 630 nm, λ(em) = 660 nm) with a high affinity constant (K = ~10(2.80) M(-1)) superior to other sugar probes. Semiempirical quantum mechanics calculations suggest that the mechanism for this high enhancement may involve the dissociation of initially nonemissive dye associates (stabilized by an intramolecular hydrogen bond) upon complex formation with sugars. The fluorescence emission of SQ-BA was also significantly enhanced in the presence of a gram-positive bacterial spore, Bacillus globigii (Bg), which serves as a simulant of B. anthracis (or anthrax) and which possesses a peptidoglycan (sugar)-rich spore coat to provide ample sites for interaction with the dye. Several peaks were observed for a pure Bg sample even with polyethyleneoxide (PEO) present in the CE separation buffer, despite the polymer's previously demonstrated ability to focus microoorganisms to a single peak during migration. Likewise, several peaks were observed for a Bg sample when capillary transient isotachophoresis (ctITP) alone was employed. However, the new combination of these techniques as "PectI" dramatically and reproducibly focused the bacteria to a single peak with no staining procedure. Using PectI, the trace detection of Bg spores (corresponding to approximately three cells per injection) along with separation efficiency enough to separate Bg from another gram-positive bacteria, Saccharomyces cerevisiae (resolution, R(s) = 6.09, and apparent plate number, N = 2.7-3.3 × 10(5)), were successfully achieved.

Spectrofluorimetric determination of etodolac, moxepril HCl and fexofenadine HCl using europium sensitized fluorescence in bulk and pharmaceutical preparations.

A simple, selective and sensitive luminescence method has been developed for the assay of etodolac (I), moxepril HCl (II) and fexofenadine HCl (III) in bulk drug and pharmaceutical formulations. The method is based on the luminescence sensitization of europium (Eu(3+)) by complexation with the studied drugs. The fluorescence intensities of the products were measured at 667 nm for (I) and at 615 for (II) and (III) while exciting at 276 for all the studied drugs. The fluorescence intensity was directly proportional to the concentration over the range (20-280), (40-240) and (30-80) ng/ml with limits of detection (LOD) = 0.93, 0.92 and 0.95 µg/ml for drugs I, II and III respectively. Optimum conditions for the formation of the complex in methanol were carefully studied. The proposed method was successfully applied for the assay of the studied drugs in pharmaceutical formulations with excellent recovery.

Manganese exposure inhibits the clearance of extracellular GABA and influences taurine homeostasis in the striatum of developing rats.

Manganese (Mn) accumulation in the brain has been shown to alter the neurochemistry of the basal ganglia. Mn-induced alterations in dopamine biology are fairly well understood, but recently more evidence has emerged characterizing the role of γ-aminobutyric acid (GABA) in this dysfunction. The purpose of this study was to determine if the previously observed Mn-induced increase in extracellular GABA (GABA(EC)) was due to altered GABA transporter (GAT) function, and whether Mn perturbs other amino acid neurotransmitters, namely taurine and glycine (known modulators of GABA). Extracellular GABA, taurine, and glycine concentrations were collected from the striatum of control (CN) or Mn-exposed Sprague-Dawley rats using in vivo microdialysis, and the GAT inhibitor nipecotic acid (NA) was used to probe GAT function. Tissue and extracellular Mn levels were significantly increased, and the Fe:Mn ratio was decreased 36-fold in the extracellular space due to Mn-exposure. NA led to a 2-fold increase in GABA(EC) of CNs, a response that was attenuated by Mn. Taurine responded inversely to GABA, and a novel 10-fold increase in taurine was observed after the removal of NA in CNs. Mn blunted this response and nearly abolished extracellular taurine throughout collection. Striatal taurine transporter (Slc6a6) mRNA levels were significantly increased with Mn-exposure, and Mn significantly increased (3)H-Taurine uptake after 3-min exposure in primary rat astrocytes. These data suggest that Mn increases GABA(EC) by inhibiting the function of GAT, and that perturbed taurine homeostasis potentially impacts neural function by jeopardizing the osmoregulatory and neuromodulatory functions of taurine in the brain.

CE-ESI-MS analysis of singly charged inorganic and organic anions using a dicationic reagent as a complexing agent.

A dicationic ion-pairing reagent, N,N'-dibutyl 1,1'-pentylenedipyrrolidinium, was used to form complexes with singly charged anions for their subsequent analysis by CE-ESI-MS in positive ion mode. This methodology offers the advantages of greater versatility and sensitivity relative to direct detection of the anions in negative ion mode, and it can be realized by a number of possible complexation strategies, including pre-column, on-column, and post-column modes. Four model anions, perfluorooctanoate, benzenesulfonate (BZSN), monochloroacetate (MCA), and trifluoromethanesulfonimide were amenable to complexation with the dicationic reagent, yielding singly charged cations with greater m/z. By optimizing various parameters, including the CE separation buffer composition and pH, the concentration of the dicationic reagent, the mode of complexation, the nebulizing gas pressure, and the sheath liquid composition, it was possible to develop a robust CE-ESI-MS method appropriate for the analysis of anions in a tap water sample. By this method, LODs were found to be 20.9 and 1.31 ng/mL for MCA and BZSN, respectively.

Extracellular norepinephrine, norepinephrine receptor and transporter protein and mRNA levels are differentially altered in the developing rat brain due to dietary iron deficiency and manganese exposure.

Manganese (Mn) is an essential trace element, but overexposure is characterized by Parkinson's like symptoms in extreme cases. Previous studies have shown that Mn accumulation is exacerbated by dietary iron deficiency (ID) and disturbances in norepinephrine (NE) have been reported. Because behaviors associated with Mn neurotoxicity are complex, the goal of this study was to examine the effects of Mn exposure and ID-associated Mn accumulation on NE uptake in synaptosomes, extracellular NE concentrations, and expression of NE transport and receptor proteins. Sprague-Dawley rats were assigned to four dietary groups: control (CN; 35 mg Fe/kg diet), iron-deficient (ID; 6 mg Fe/kg diet), CN with Mn exposure (via the drinking water; 1 g Mn/L) (CNMn), and ID with Mn (IDMn). (3)H-NE uptake decreased significantly (R=-0.753, p=0.001) with increased Mn concentration in the locus coeruleus, while decreased Fe was associated with decreased uptake of (3)H-NE in the caudate putamen (R=0.436, p=0.033) and locus coeruleus (R=0.86; p<0.001). Extracellular concentrations of NE in the caudate putamen were significantly decreased in response to Mn exposure and ID (p<0.001). A diverse response of Mn exposure and ID was observed on mRNA and protein expression of NE transporter (NET) and alpha(2) adrenergic receptor. For example, elevated brain Mn and decreased Fe caused an approximate 50% decrease in NET and alpha(2) adrenergic receptor protein expression in several brain regions, with reductions in mRNA expression also observed. These data suggest that Mn exposure results in a decrease in NE uptake and extracellular NE concentrations via altered expression of transport and receptor proteins.

Manganese exposure alters extracellular GABA, GABA receptor and transporter protein and mRNA levels in the developing rat brain.

Unlike other essential trace elements (e.g., zinc and iron) it is the toxicity of manganese (Mn) that is more common in human populations than its deficiency. Data suggest alterations in dopamine biology may drive the effects associated with Mn neurotoxicity, though recently gamma-aminobutyric acid (GABA) has been implicated. In addition, iron deficiency (ID), a common nutritional problem, may cause disturbances in neurochemistry by facilitating accumulation of Mn in the brain. Previous data from our lab have shown decreased brain tissue levels of GABA as well as decreased (3)H-GABA uptake in synaptosomes as a result of Mn exposure and ID. These results indicate a possible increase in the concentration of extracellular GABA due to alterations in expression of GABA transport and receptor proteins. In this study weanling-male Sprague-Dawley rats were randomly placed into one of four dietary treatment groups: control (CN; 35mg Fe/kg diet), iron-deficient (ID; 6mg Fe/kg diet), CN with Mn supplementation (via the drinking water; 1g Mn/l) (CNMn), and ID with Mn supplementation (IDMn). Using in vivo microdialysis, an increase in extracellular GABA concentrations in the striatum was observed in response to Mn exposure and ID although correlational analysis reveals that extracellular GABA is related more to extracellular iron levels and not Mn. A diverse effect of Mn exposure and ID was observed in the regions examined via Western blot and RT-PCR analysis, with effects on mRNA and protein expression of GAT-1, GABA(A), and GABA(B) differing between and within the regions examined. For example, Mn exposure reduced GAT-1 protein expression by approximately 50% in the substantia nigra, while increasing mRNA expression approximately four-fold, while in the caudate putamen mRNA expression was decreased with no effect on protein expression. These data suggest that Mn exposure results in an increase in extracellular GABA concentrations via altered expression of transport and receptor proteins, which may be the basis of the neurological characteristics of manganism.