The Anti-Cancer Activity of the Naturally Occurring Dipeptide Carnosine: Potential for Breast Cancer.

Carnosine is an endogenous dipeptide composed of ß-alanine and L-histidine, possessing a multimodal pharmacodynamic profile that includes anti-inflammatory and anti-oxidant activities. Carnosine has also shown its ability to modulate cell proliferation, cell cycle arrest, apoptosis, and even glycolytic energy metabolism, all processes playing a key role in the context of cancer. Cancer is one of the most dreaded diseases of the 20th and 21st centuries. Among the different types of cancer, breast cancer represents the most common non-skin cancer among women, accounting for an estimated 15% of all cancer-related deaths in women. The main aim of the present review was to provide an overview of studies on the anti-cancer activity of carnosine, and in particular its activity against breast cancer. We also highlighted the possible advantages and limitations involved in the use of this dipeptide. The first part of the review entailed a brief description of carnosine's biological activities and the pathophysiology of cancer, with a focus on breast cancer. The second part of the review described the anti-tumoral activity of carnosine, for which numerous studies have been carried out, especially at the preclinical level, showing promising results. However, only a few studies have investigated the therapeutic potential of this dipeptide for breast cancer prevention or treatment. In this context, carnosine has shown to be able to decrease the size of cancer cells and their viability. It also reduces the levels of vascular endothelial growth factor (VEGF), cyclin D1, NAD+, and ATP, as well as cytochrome c oxidase activity in vitro. When tested in mice with induced breast cancer, carnosine proved to be non-toxic to healthy cells and exhibited chemopreventive activity by reducing tumor growth. Some evidence has also been reported at the clinical level. A randomized phase III prospective placebo-controlled trial showed the ability of Zn-carnosine to prevent dysphagia in breast cancer patients undergoing adjuvant radiotherapy. Despite this evidence, more preclinical and clinical studies are needed to better understand carnosine's anti-tumoral activity, especially in the context of breast cancer.

Lung Surfactant Decreases Biochemical Alterations and Oxidative Stress Induced by a Sub-Toxic Concentration of Carbon Nanoparticles in Alveolar Epithelial and Microglial Cells.

Carbon-based nanomaterials are nowadays attracting lots of attention, in particular in the biomedical field, where they find a wide spectrum of applications, including, just to name a few, the drug delivery to specific tumor cells and the improvement of non-invasive imaging methods. Nanoparticles inhaled during breathing accumulate in the lung alveoli, where they interact and are covered with lung surfactants. We recently demonstrated that an apparently non-toxic concentration of engineered carbon nanodiamonds (ECNs) is able to induce oxidative/nitrosative stress, imbalance of energy metabolism, and mitochondrial dysfunction in microglial and alveolar basal epithelial cells. Therefore, the complete understanding of their "real" biosafety, along with their possible combination with other molecules mimicking the in vivo milieu, possibly allowing the modulation of their side effects becomes of utmost importance. Based on the above, the focus of the present work was to investigate whether the cellular alterations induced by an apparently non-toxic concentration of ECNs could be counteracted by their incorporation into a synthetic lung surfactant (DPPC:POPG in 7:3 molar ratio). By using two different cell lines (alveolar (A549) and microglial (BV-2)), we were able to show that the presence of lung surfactant decreased the production of ECNs-induced nitric oxide, total reactive oxygen species, and malondialdehyde, as well as counteracted reduced glutathione depletion (A549 cells only), ameliorated cell energy status (ATP and total pool of nicotinic coenzymes), and improved mitochondrial phosphorylating capacity. Overall, our results on alveolar basal epithelial and microglial cell lines clearly depict the benefits coming from the incorporation of carbon nanoparticles into a lung surfactant (mimicking its in vivo lipid composition), creating the basis for the investigation of this combination in vivo.

Biological applications of microchip electrophoresis with amperometric detection: in vivo monitoring and cell analysis.

Microchip electrophoresis with amperometric detection (ME-EC) is a useful tool for the determination of redox active compounds in complex biological samples. In this review, a brief background on the principles of ME-EC is provided, including substrate types, electrode materials, and electrode configurations. Several different detection approaches are described, including dual-channel systems for dual-electrode detection and electrochemistry coupled with fluorescence and chemiluminescence. The application of ME-EC to the determination of catecholamines, adenosine and its metabolites, and reactive nitrogen and oxygen species in microdialysis samples and cell lysates is also detailed. Lastly, approaches for coupling of ME-EC with microdialysis sampling to create separation-based sensors that can be used for near real-time monitoring of drug metabolism and neurotransmitters in freely roaming animals are provided. Graphical abstract.

Modulation of Pro-Oxidant and Pro-Inflammatory Activities of M1 Macrophages by the Natural Dipeptide Carnosine.

Carnosine is a natural endogenous dipeptide widely distributed in mammalian tissues, existing at particularly high concentrations in the muscles and brain and possesses well-characterized antioxidant and anti-inflammatory activities. In an in vitro model of macrophage activation, induced by lipopolysaccharide + interferon-gamma (LPS + IFN-γ), we here report the ability of carnosine to modulate pro-oxidant and pro-inflammatory activities of macrophages, representing the primary cell type that is activated as a part of the immune response. An ample set of parameters aimed to evaluate cytotoxicity (MTT assay), energy metabolism (HPLC), gene expressions (high-throughput real-time PCR (qRT-PCR)), protein expressions (western blot) and nitric oxide production (qRT-PCR and HPLC), was used to assess the effects of carnosine on activated macrophages challenged with a non cytotoxic LPS (100 ng/mL) + IFN-γ (600 U/mL) concentration. In our experimental model, main carnosine beneficial effects were: (1) the modulation of nitric oxide production and metabolism; (2) the amelioration of the macrophage energy state; (3) the decrease of the expressions of pro-oxidant enzymes (Nox-2, Cox-2) and of the lipid peroxidation product malondialdehyde; (4) the restoration and/or increase of the expressions of antioxidant enzymes (Gpx1, SOD-2 and Cat); (5) the increase of the transforming growth factor-ß1 (TGF-ß1) and the down-regulation of the expressions of interleukins 1ß and 6 (IL-1ß and IL-6) and 6) the increase of the expressions of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1). According to these results carnosine is worth being tested in the treatment of diseases characterized by elevated levels of oxidative stress and inflammation (atherosclerosis, cancer, depression, metabolic syndrome, and neurodegenerative diseases).

Evaluation of dual electrode configurations for microchip electrophoresis used for voltammetric characterization of electroactive species.

Microchip electrophoresis coupled with amperometric detection is more popular than voltammetric detection due to the lower limits of detection that can be achieved. However, voltammetry provides additional information about the redox properties of the analyte that can be used for peak identification. In this paper, two dual electrode configurations for microchip electrophoresis are described and evaluated for obtaining voltammetric information using amperometry. The dual-series electrode configuration was first evaluated to generate current ratios in a single run by applying two different potentials to the working electrodes placed perpendicular to the separation channel. However, it was found that it is difficult to obtain realistic current ratios with this configuration, primarily due to the relative placement of electrodes with respect to the channel end of the simple-t microchip. Correction factors were needed to obtain current ratios similar to those that would be obtained for sequential injections at two different potentials using a single electrode. A second approach using a dual-channel chip with two parallel electrodes was then developed and evaluated for obtaining voltammetric identification. The newly developed microchip permitted the injection of same amount of sample into two unique separation channels, each with an electrode at a different detection potential. Migration times and current ratios for several biologically important molecules and potential interferences including nitrite, tyrosine, hydrogen peroxide, and azide were obtained and compared to the responses obtained for analytes found in macrophage cell lysates.

Sub-Toxic Human Amylin Fragment Concentrations Promote the Survival and Proliferation of SH-SY5Y Cells via the Release of VEGF and HspB5 from Endothelial RBE4 Cells.

Human amylin is a 37-residue peptide hormone (hA1-37) secreted by ß-cells of the pancreas and, along with insulin, is directly associated with type 2 diabetes mellitus (T2DM). Amyloid deposits within the islets of the pancreas represent a hallmark of T2DM. Additionally, amylin aggregates have been found in blood vessels and/or brain of patients with Alzheimer's disease, alone or co-deposited with ß-amyloid. The purpose of this study was to investigate the neuroprotective potential of human amylin in the context of endothelial-neuronal "cross-talk". We initially performed dose-response experiments to examine cellular toxicity (quantified by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] MTT assay) of different hA17⁻29 concentrations in endothelial cells (RBE4). In the culture medium of these cells, we also measured heat shock protein B5 (HspB5) levels by ELISA, finding that even a sub-toxic concentration of hA17⁻29 (3 µM) produced an increase of HspB5. Using a cell medium of untreated and RBE4 challenged for 48 h with a sub-toxic concentration of hA17⁻29, we determined the potential beneficial effect of their addition to the medium of neuroblastoma SH-SY5Y cells. These cells were subsequently incubated for 48 h with a toxic concentration of hA17⁻29 (20 µM). We found a complete inhibition of hA17⁻29 toxicity, potentially related to the presence in the conditioned medium not only of HspB5, but also of vascular endothelial growth factor (VEGF). Pre-treating SH-SY5Y cells with the anti-Flk1 antibody, blocking the VEGF receptor 2 (VEGFR2), significantly decreased the protective effects of the conditioned RBE4 medium. These data, obtained by indirectly measuring VEGF activity, were strongly corroborated by the direct measurement of VEGF levels in conditioned RBE4 media as detected by ELISA. Altogether, these findings highlighted a novel role of sub-toxic concentrations of human amylin in promoting the secretion of proteic factors by endothelial cells (HspB5 and VEGF) that support the survival and proliferation of neuron-like cells.

Non-toxic engineered carbon nanodiamond concentrations induce oxidative/nitrosative stress, imbalance of energy metabolism, and mitochondrial dysfunction in microglial and alveolar basal epithelial cells.

Engineered nanoparticles are finding a wide spectrum of biomedical applications, including drug delivery and capacity to trigger cytotoxic phenomena, potentially useful against tumor cells. The full understanding of their biosafety and interactions with cell processes is mandatory. Using microglial (BV-2) and alveolar basal epithelial (A549) cells, in this study we determined the effects of engineered carbon nanodiamonds (ECNs) on cell viability, nitric oxide (NO) and reactive oxygen species (ROS) production, as well as on energy metabolism. Particularly, we initially measured decrease in cell viability as a function of increasing ECNs doses, finding similar cytotoxic ECN effects in the two cell lines. Subsequently, using apparently non-cytotoxic ECN concentrations (2 µg/mL causing decrease in cell number < 5%) we determined NO and ROS production, and measured the concentrations of compounds related to energy metabolism, mitochondrial functions, oxido-reductive reactions, and antioxidant defences. We found that in both cell lines non-cytotoxic ECN concentrations increased NO and ROS production with sustained oxidative/nitrosative stress, and caused energy metabolism imbalance (decrease in high energy phosphates and nicotinic coenzymes) and mitochondrial malfunctioning (decrease in ATP/ADP ratio).These results underline the importance to deeply investigate the molecular and biochemical changes occurring upon the interaction of ECNs (and nanoparticles in general) with living cells, even at apparently non-toxic concentration. Since the use of ECNs in biomedical field is attracting increasing attention the complete evaluation of their biosafety, toxicity and/or possible side effects both in vitro and in vivo is mandatory before these highly promising tools might find the correct application.

Continuous monitoring of adenosine and its metabolites using microdialysis coupled to microchip electrophoresis with amperometric detection.

Rapid monitoring of concentration changes of neurotransmitters and energy metabolites is important for understanding the biochemistry of neurological disease as well as for developing therapeutic options. This paper describes the development of a separation-based sensor using microchip electrophoresis (ME) with electrochemical (EC) detection coupled to microdialysis (MD) sampling for continuous on-line monitoring of adenosine and its downstream metabolites. The device was fabricated completely in PDMS. End-channel electrochemical detection was accomplished using a carbon fiber working electrode embedded in the PDMS. The separation conditions for adenosine, inosine, hypoxanthine, and guanosine were investigated using a ME-EC chip with a 5-cm long separation channel. The best resolution was achieved using a background electrolyte consisting of 35 mM sodium borate at pH 10, 15% dimethyl sulfoxide (DMSO), and 2 mM sodium dodecyl sulphate (SDS), and a field strength of 222 V/cm. Under these conditions, all four purines were separated in less than 85 s. Using a working electrode detection potential of 1.4 vs Ag/AgCl, the limits of detection were 25, 33, 10, and 25 µM for adenosine, inosine, hypoxanthine, and guanosine, respectively. The ME-EC chip was then coupled to microdialysis sampling using a novel all-PDMS microdialysis-microchip interface that was reversibly sealed. This made alignment of the working electrode with the end of the separation channel much easier and more reproducible than could be obtained with previous MD-ME-EC systems. The integrated device was then used to monitor the enzymatic conversion of adenosine to inosine in vitro.

Microchip electrophoresis with laser-induced fluorescence detection for the determination of the ratio of nitric oxide to superoxide production in macrophages during inflammation.

It is well known that excessive production of reactive oxygen and nitrogen species is linked to the development of oxidative stress-driven disorders. In particular, nitric oxide (NO) and superoxide (O2•-) play critical roles in many physiological and pathological processes. This article reports the use of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate and MitoSOX Red in conjunction with microchip electrophoresis and laser-induced fluorescence detection for the simultaneous detection of NO and O2•- in RAW 264.7 macrophage cell lysates following different stimulation procedures. Cell stimulations were performed in the presence and absence of cytosolic (diethyldithiocarbamate) and mitochondrial (2-methoxyestradiol) superoxide dismutase (SOD) inhibitors. The NO/O2•- ratios in macrophage cell lysates under physiological and proinflammatory conditions were determined. The NO/O2•- ratios were 0.60 ± 0.07 for unstimulated cells pretreated with SOD inhibitors, 1.08 ± 0.06 for unstimulated cells in the absence of SOD inhibitors, and 3.14 ± 0.13 for stimulated cells. The effect of carnosine (antioxidant) or Ca2+ (intracellular messenger) on the NO/O2•- ratio was also investigated. Graphical Abstract Simultaneous detection of nitric oxide and superoxide in macrophage cell lysates.

Carnosine modulates nitric oxide in stimulated murine RAW 264.7 macrophages.

Excess nitric oxide (NO) production occurs in several pathological states, including neurodegeneration, ischemia, and inflammation, and is generally accompanied by increased oxidative/nitrosative stress. Carnosine [ß-alanine-histidine (ß-Ala-His)] has been reported to decrease oxidative/nitrosative stress-associated cell damage by reducing the amount of NO produced. In this study, we evaluated the effect of carnosine on NO production by murine RAW 264.7 macrophages stimulated with lipopolysaccharides + interferon-γ. Intracellular NO and intracellular and extracellular nitrite were measured by microchip electrophoresis with laser-induced fluorescence and by the Griess assay, respectively. Results showed that carnosine causes an apparent suppression of total NO production by stimulated macrophages accompanied by an unexpected simultaneous drastic increase in its intracellular low toxicity endproduct, nitrite, with no inhibition of inducible nitric oxide synthase (iNOS). ESI-MS and NMR spectroscopy in a cell-free system showed the formation of multiple adducts (at different ratios) of carnosine-NO and carnosine-nitrite, involving both constituent amino acids (ß-Ala and His) of carnosine, thus providing a possible mechanism for the changes in free NO and nitrite in the presence of carnosine. In stimulated macrophages, the addition of carnosine was also characterized by changes in the expression of macrophage activation markers and a decrease in the release of IL-6, suggesting that carnosine might alter M1/M2 macrophage ratio. These results provide evidence for previously unknown properties of carnosine that modulate the NO/nitrite ratio of stimulated macrophages. This modulation is also accompanied by changes in the release of pro-inflammatory molecules, and does not involve the inhibition of iNOS activity.

Receptor-mediated toxicity of human amylin fragment aggregated by short- and long-term incubations with copper ions.

Human amylin (hA1-37) is a polypeptide hormone secreted in conjunction with insulin from the pancreatic ß-cells involved in the pathogenesis of type 2 diabetes mellitus (T2DM). The shorter fragment hA17-29 than full-length peptide is capable to form amyloids "in vitro". Here, we monitored the time course of hA17-29 ß-amyloid fibril and oligomer formation [without and with copper(II)], cellular toxicity of different amyloid aggregates, and involvement of specific receptors (receptor for advanced glycation end-products, RAGE; low-affinity nerve growth factor receptor, p75-NGFR) in aggregate toxicity. Fibril and oligomer formation of hA17-29 incubated at 37 °C for 0, 48, and 120 h, without or with copper(II), were measured by the thioflavin T fluorescence assay and ELISA, respectively. Toxicity of hA17-29 aggregates and effects of anti-RAGE and anti-p75-NGFR antibodies were evaluated on neuroblastoma SH-SY5Y viability. Fluorescence assay of hA17-29 indicates an initial slow rate of soluble fibril formation (48 h), followed by a slower rate of insoluble aggregate formation (120 h). The highest quantity of oligomers was recorded when hA17-29 was pre-aggregated for 48 h in the presence of copper(II) showing also the maximal cell toxicity (-44% of cell viability, p < 0.01 compared to controls). Anti-RAGE or anti-p75-NGFR antibodies almost abolished cell toxicity of hA17-29 aggregates. These results indicate that copper(II) influences the aggregation process and hA17-29 toxicities are especially attributable to oligomeric aggregates. hA17-29 aggregate toxicity seems to be mediated by RAGE and p75-NGFR receptors which might be potential targets for new drugs in T2DM treatment.

Microchip electrophoresis with amperometric detection method for profiling cellular nitrosative stress markers.

The overproduction of nitric oxide (NO) in cells results in nitrosative stress due to the generation of highly reactive species such as peroxynitrite and N2O3. These species disrupt the cellular redox processes through the oxidation, nitration, and nitrosylation of important biomolecules. Microchip electrophoresis (ME) is a fast separation method that can be used to profile cellular nitrosative stress through the separation of NO and nitrite from other redox-active intracellular components such as cellular antioxidants. This paper describes a ME method with electrochemical detection (ME-EC) for the separation of intracellular nitrosative stress markers in macrophage cells. The separation of nitrite, azide (interference), iodide (internal standard), tyrosine, glutathione, and hydrogen peroxide (neutral marker) was achieved in under 40 s using a run buffer consisting of 7.5 to 10 mM NaCl, 10 mM boric acid, and 2 mM TTAC at pH 10.3 to 10.7. Initially, NO production was monitored by the detection of nitrite (NO2(-)) in cell lysates. There was a 2.5- to 4-fold increase in NO2(-) production in lipopolysaccharide (LPS)-stimulated cells. The concentration of NO2(-) inside a single unstimulated macrophage cell was estimated to be 1.41 mM using the method of standard additions. ME-EC was then used for the direct detection of NO and glutathione in stimulated and native macrophage cell lysates. NO was identified in these studies based on its migration time and rapid degradation kinetics. The intracellular levels of glutathione in native and stimulated macrophages were also compared, and no significant difference was observed between the two conditions.

An integrated microfluidic device for monitoring changes in nitric oxide production in single T-lymphocyte (Jurkat) cells.

A considerable amount of attention has been focused on the analysis of single cells in an effort to better understand cell heterogeneity in cancer and neurodegenerative diseases. Although microfluidic devices have several advantages for single cell analysis, few papers have actually demonstrated the ability of these devices to monitor chemical changes in perturbed biological systems. In this paper, a new microfluidic channel manifold is described that integrates cell transport, lysis, injection, electrophoretic separation, and fluorescence detection into a single device, making it possible to analyze individual cells at a rate of 10 cells/min in an automated fashion. The system was employed to measure nitric oxide (NO) production in single T-lymphocytes (Jurkat cells) using a fluorescent marker, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA). The cells were also labeled with 6-carboxyfluorescein diacetate (6-CFDA) as an internal standard. The NO production by control cells was compared to that of cells stimulated using lipopolysaccharide (LPS), which is known to cause the expression of inducible nitric oxide synthase (iNOS) in immune-type cells. Statistical analysis of the resulting electropherograms from a population of cells indicated a 2-fold increase in NO production in the induced cells. These results compare nicely to a recently published bulk cell analysis of NO.

In-channel amperometric detection for microchip electrophoresis using a wireless isolated potentiostat.

The combination of microchip electrophoresis with amperometric detection leads to a number of analytical challenges that are associated with isolating the detector from the high voltages used for the separation. While methods such as end-channel alignment and the use of decouplers have been employed, they have limitations. A less common method has been to utilize an electrically isolated potentiostat. This approach allows placement of the working electrode directly in the separation channel without using a decoupler. This paper explores the use of microchip electrophoresis and electrochemical detection with an electrically isolated potentiostat for the separation and in-channel detection of several biologically important anions. The separation employed negative polarity voltages and tetradecyltrimethylammonium bromide (as a buffer modifier) for the separation of nitrite (NO2⁻), glutathione, ascorbic acid, and tyrosine. A half-wave potential shift of approximately negative 500 mV was observed for NO2⁻ and H2O2 standards in the in-channel configuration compared to end-channel. Higher separation efficiencies were observed for both NO2⁻ and H2O2 with the in-channel detection configuration. The limits of detection were approximately two-fold lower and the sensitivity was approximately two-fold higher for in-channel detection of nitrite when compared to end-channel. The application of this microfluidic device for the separation and detection of biomarkers related to oxidative stress is described.

A microchip electrophoresis device with on-line microdialysis sampling and on-chip sample derivatization by naphthalene 2,3-dicarboxaldehyde/2-mercaptoethanol for amino acid and peptide analysis.

The integration of rapid on-chip sample derivatization employing naphthalene 2,3-dicarboxaldehyde and 2-mercaptoethanol (NDA/2ME) with an easily assembled microdialysis/microchip electrophoresis device was carried out. The microchip device consisted of a glass layer with etched microfluidic channels that was sealed with a layer of poly(dimethylsiloxane) (PDMS) via plasma oxidation. This simple sealing procedure alleviated the need for glass thermal bonding and allowed the device to be re-sealed in the event of blockages within the channels. The device was used for analysis of a mixture of amino acids and peptides derivatized on-chip with NDA/2ME for laser-induced fluorescence (LIF) detection. A 0.6 mM NDA/1.2 mM 2ME mixture was simply added into the buffer reservoir for dynamic on-column derivatization of sample mixtures introduced at a flow rate of 1.0 microl/min. Using this scheme, sample injection plugs were derivatized and separated simultaneously. Injections of ca. 12 fmol of 5 mM amino acid and peptide samples were conducted using the system. Finally, a three-component mixture of Arg, Gly-Pro, and Asp was sampled from a vial using microdialysis, derivatized, separated and detected with the system. The ultimate goal of this effort is the creation of a micro-total analysis system for high-temporal resolution monitoring of primary amines in biological systems.

Microchip capillary electrophoresis: application to peptide analysis.

The development of analytical methodologies to elucidate mechanisms of peptide transport and metabolism is important for the understanding of disease states and the design of effective drug therapies. Interest in the use of microchip capillary electrophoresis (CE) devices for peptide analysis stems from the ability to perform fast, highly efficient separations combined with small sample volume requirements. Many of the separation modes developed on conventional systems, including electrochromatography, isoelectric focusing, and electrophoretic bioaffinity assays, have been demonstrated on microchip devices. Steps that include sample preparation and labeling can also be integrated onto the microchip platform. This chapter will discuss considerations for peptide analysis using microchip CE and will focus on different approaches to sample preparation, separation, and detection.

Comparison of the performance characteristics of poly(dimethylsiloxane) and Pyrex microchip electrophoresis devices for peptide separations.

A comparative study of electrophoretic separations of fluorescently labeled peptides and amino acids on poly(dimethylsiloxane) (PDMS) and Pyrex microchips is presented. The separation parameters for each microchip substrate were compared, including electroosmotic flow, plate numbers, resolution, and limits of detection. The effect of buffer composition on the separation was also investigated. Acceptable separations were obtained for most peptides with both substrates; however, PDMS chips exhibited much lower separation efficiencies and longer analysis times.

Amino acid and peptide analysis using derivatization with p-nitrophenol-2,5-dihydroxyphenylacetate bis-tetrahydropyranyl ether and capillary electrophoresis with electrochemical detection.

The amine derivatization reagent p-nitrophenol-2,5-dihydroxyphenylacetate bis-tetrahydropyranyl ether (NDTE) was used in conjunction with capillary electrophoresis (CE) and electrochemical detection (EC) for the pre-separation derivatization of primary amine analytes present in aqueous solution. Glycine, several dipeptides and angiotensin II were used as model analytes. A miniaturized EC detection cell was designed and fabricated, which featured a fractured-joint field decoupler with a fixed end-column carbon fiber electrode. When a series of glycine and angiotensin II calibration solutions were derivatized with NDTE followed by CE-EC determination, linear calibration plots resulted with pre-derivatization concentration limits of detection of 500 nM (106 attomoles on-column) and 6 microM (1.275 femtomoles on-column), respectively.

Detection of homocysteine by conventional and microchip capillary electrophoresis/electrochemistry.

A method based on capillary electrophoresis (CE) with electrochemical (EC) detection for the determination of both total homocysteine (tHcy) and protein-bound homocysteine (pbHcy) in plasma is described. Both end-column and off-column amperometric detection were investigated. Off-column detection resulted in a more sensitive assay for the determination of homocysteine (Hcy). The detection limit for homocysteine was 500 nM using off-column EC detection and the response was linear over the range 1-100 microM. Therefore, this assay is appropriate for the quantification of Hcy over the physiological concentration ranges found in all disease states. Methodologies for the determination of tHcy and pbHcy in human plasma were investigated and optimized and the concentrations of both pbHcy and tHcy in plasma obtained from a healthy individual were determined to be 2.79+/-0.31 nuM (n = 4) and 3.37+/-0.15 microM (n = 3), respectively. The methodology was then transferred to a microchip CE-EC format and Hcy and reduced glutathione (GSH) were detected. Future work will focus on the development of ancillary methodologies to identify the other forms of Hcy in vivo.