Copper accumulation in senescent cells: Interplay between copper transporters and impaired autophagy.

Cellular senescence is characterized by irreversible growth arrest incurred through either replicative exhaustion or by pro-oncogenic cellular stressors (radioactivity, oxidative stress, oncogenic activation). The enrichment of senescent cells in tissues with age has been associated with tissue dyshomeostasis and age-related pathologies including cancers, neurodegenerative disorders (e.g. Alzheimer's, Parkinson's, etc.) and metabolic disorders (e.g. diabetes). We identified copper accumulation as being a universal feature of senescent cells [mouse embryonic fibroblasts (MEF), human prostate epithelial cells and human diploid fibroblasts] in vitro. Elevated copper in senescent MEFs was accompanied by elevated levels of high-affinity copper uptake protein 1 (Ctr1), diminished levels of copper-transporting ATPase 1 (Atp7a) (copper export) and enhanced antioxidant defence reflected by elevated levels of glutathione (GSH), superoxide dismutase 1 (SOD1) and glutaredoxin 1 (Grx1). The levels of intracellular copper were further increased in senescent MEFs cultured in copper supplemented medium and in senescent Mottled Brindled (Mobr) MEFs lacking functional Atp7a. Finally, we demonstrated that the restoration/preservation of autophagic-lysosomal degradation in senescent MEFs following rapamycin treatment correlated with attenuation of copper accumulation in these cells despite a further decrease in Atp7a levels. This study for the first time establishes a link between Atp7a and the autophagic-lysosomal pathway, and a requirement for both to effect efficient copper export. Such a connection between cellular autophagy and copper homeostasis is significant, as both have emerged as important facets of age-associated degenerative disease.

N-acetylcysteine modulates glutamatergic dysfunction and depressive behavior in Huntington's disease.

Glutamatergic dysfunction has been implicated in the pathogenesis of depressive disorders and Huntington's disease (HD), in which depression is the most common psychiatric symptom. Synaptic glutamate homeostasis is regulated by cystine-dependent glutamate transporters, including GLT-1 and system xc- In HD, the enzyme regulating cysteine (and subsequently cystine) production, cystathionine-γ-lygase, has recently been shown to be lowered. The aim of the present study was to establish whether cysteine supplementation, using N-acetylcysteine (NAC) could ameliorate glutamate pathology through the cystine-dependent transporters, system xc- and GLT-1. We demonstrate that the R6/1 transgenic mouse model of HD has lower basal levels of cystine, and showed depressive-like behaviors in the forced-swim test. Administration of NAC reversed these behaviors. This effect was blocked by co-administration of the system xc- and GLT-1 inhibitors CPG and DHK, showing that glutamate transporter activity was required for the antidepressant effects of NAC. NAC was also able to specifically increase glutamate in HD mice, in a glutamate transporter-dependent manner. These in vivo changes reflect changes in glutamate transporter protein in HD mice and human HD post-mortem tissue. Furthermore, NAC was able to rescue changes in key glutamate receptor proteins related to excitotoxicity in HD, including NMDAR2B. Thus, we have shown that baseline reductions in cysteine underlie glutamatergic dysfunction and depressive-like behavior in HD and these changes can be rescued by treatment with NAC. These findings have implications for the development of new therapeutic approaches for depressive disorders.

Blind column selection protocol for two-dimensional high performance liquid chromatography.

The selection of two orthogonal columns for two-dimensional high performance liquid chromatography (LC×LC) separation of natural product extracts can be a labour intensive and time consuming process and in many cases is an entirely trial-and-error approach. This paper introduces a blind optimisation method for column selection of a black box of constituent components. A data processing pipeline, created in the open source application OpenMS®, was developed to map the components within the mixture of equal mass across a library of HPLC columns; LC×LC separation space utilisation was compared by measuring the fractional surface coverage, fcoverage. It was found that for a test mixture from an opium poppy (Papaver somniferum) extract, the combination of diphenyl and C18 stationary phases provided a predicted fcoverage of 0.48 and was matched with an actual usage of 0.43. OpenMS®, in conjunction with algorithms designed in house, have allowed for a significantly quicker selection of two orthogonal columns, which have been optimised for a LC×LC separation of crude extractions of plant material.

Comprehensive sample analysis using high performance liquid chromatography with multi-detection.

Herein we assess the separation space offered by a liquid chromatography system with an optimised uni-dimensional separation for the determination of the key chemical entities in the highly complex matrix of a tobacco leaf extract. Multiple modes of detection, including UV-visible absorbance, chemiluminescence (acidic potassium permanganate, manganese(IV), and tris(2,2'-bipyridine)ruthenium(III)), mass spectrometry and DPPH radical scavenging were used in an attempt to systematically reduce the data complexity of the sample whilst obtaining a greater degree of molecule-specific information. A large amount of chemical data was obtained, but several limitations in the ability to assign detector responses to particular compounds, even with the aid of complementary detection systems, were observed. Thirty-three compounds were detected via MS on the tobacco extract and 12 out of 32 compounds gave a peak height ratio (PHR) greater than 0.33 on one or more detectors. This paper serves as a case study of these limitations, illustrating why multidimensional chromatography is an important consideration when developing a comprehensive chemical detection system.

Chemiluminescence detection of heroin in illicit drug samples.

Heroin (3,6-diacetylmorphine) and several important extraction and synthesis impurities (morphine, 6-monoacetylmorphine, codeine and 6-acetylcodeine) were determined in illicit drug samples, using high performance liquid chromatography with 'parallel segmented flow', which enabled the simultaneous use of three complementary modes of detection (UV-absorbance, tris(2,2'-bipyridine)ruthenium(III) chemiluminescence and permanganate chemiluminescence). This rapid and sensitive approach for the analysis of street heroin was used to explore the chemistry of a proposed heroin screening test that is based on the relative response with these two chemiluminescence reagents using flow injection analysis. Although heroin was the major constituent of the six drug samples (between 16% and 67% by mass), the synthetic by-product 6-acetylcodeine (2.5-8.3%) made a greater contribution to the total [Ru(bipy)3](3+) chemiluminescence response of the screening test. The signal with permanganate was primarily due to the presence of 6-monoacetylmorphine (0.9-29%), and was therefore indicative of the degree of sample degradation during clandestine manufacture or poor storage conditions prior to the drug seizure. In the second part of the screening test, the sample is treated with sodium hydroxide, which results in a large increase in the signal with permanganate, due to the rapid hydrolysis of heroin to 6-monoacetylmorphine. As the emission of these two reagents with morphinan-alkaloids and their derivatives largely depends on the substituent at the O(3) position, the slower hydrolysis of 6-monoacetylmorphine to morphine, and 6-acetylcodeine to codeine, did not have a major impact on the characteristic pattern of responses in the screening test.

Autocatalytic chemiluminescence sheds new light on the classic permanganate-oxalate reaction.

The emission of light from the permanganate-oxalate reaction enables monitoring of intermediates not accessible through traditional spectrophotometric interrogation. Despite the inherent complexity of the underlying chemical reactions and equilibria, the emission intensity-time profile was characterized by a simple model combining previously independent minimalistic descriptions of chemiluminescence and autocatalysis. The generation of the electronically excited [Mn(II)]* emitter and the acceleration of the reaction even in the presence of high initial concentrations of Mn(II) (under conditions that preclude accumulation of colloidal Mn(IV)) provide new evidence for the reduction of manganese species by a reactive radical intermediate as a supplementary positive feedback loop to the formation of Mn(II).

Screening for antioxidants in complex matrices using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection.

The use of high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection to screen for antioxidants in complex plant-derived samples was evaluated in comparison with two conventional post-column radical scavenging assays (2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS(+))). In this approach, acidic potassium permanganate can react with readily oxidisable compounds (potential antioxidants), post-column, to produce chemiluminescence. Using flow injection analysis, experimental parameters that afforded the most suitable permanganate chemiluminescence signal for a range of known antioxidants were studied in a univariate approach. Optimum conditions were found to be: 1×10(-3)M potassium permanganate solution containing 1% (w/v) sodium polyphosphates adjusted to pH 2 with sulphuric acid, delivered at a flow rate of 2.5 mL min(-1) per line. Further investigations showed some differences in detection selectivity between HPLC with the optimised post-column permanganate chemiluminescence detection and DPPH and ABTS(+) assays towards antioxidant standards. However, permanganate chemiluminescence detection was more sensitive. Moreover, screening for antioxidants in green tea, cranberry juice and thyme using potassium permanganate chemiluminescence offers several advantages over the traditional DPPH and ABTS(+) assays, such as faster reagent preparation and superior stability; simpler post-column reaction manifold; and greater compatibility with fast chromatographic separations using monolithic columns.

High-performance liquid chromatography with post-column 2,2'-diphenyl-1-picrylhydrazyl radical scavenging assay: methodological considerations and application to complex samples.

An improved post-column 2,2'-diphenyl-1-picrylhydrazyl (DPPH*) radical scavenging assay for the screening of antioxidants in complex matrices was developed. Experimental parameters believed to be influential to DPPH* response were studied in a univariate approach. Optimum conditions were found to be: 5 x 10(-5) M DPPH* reagent prepared in a 75% methanol: 25% 40 mM citric acid-sodium citrate buffer (pH 6) solution, degassed with nitrogen; reaction coil of 2 m x 0.25 mm i.d. PEEK tubing; detection at 521 nm; analysis at room temperature. The analytical utility of this protocol was evaluated by screening for antioxidants in thyme and green tea, in comparison with two commonly employed methodologies.

The assessment of pi-pi selective stationary phases for two-dimensional HPLC analysis of foods: application to the analysis of coffee.

Differences between alkyl, dipole-dipole, hydrogen bonding, and pi-pi selective surfaces represented by non-resonance and resonance pi-stationary phases have been assessed for the separation of 'Ristretto' café espresso by employing 2DHPLC techniques with C18 phase selectivity detection. Geometric approach to factor analysis (GAFA) was used to measure the detected peaks (N), spreading angle (beta), correlation, practical peak capacity (n(p)) and percentage usage of the separations space, as an assessment of selectivity differences between regional quadrants of the two-dimensional separation plane. Although all tested systems were correlated to some degree to the C18 dimension, regional measurement of separation divergence revealed that performance of specific systems was better for certain sample components. The results illustrate that because of the complexity of the 'real' sample obtaining a truly orthogonal two-dimensional system for complex samples of natural origin may be practically impossible.

High performance liquid chromatography with two simultaneous on-line antioxidant assays: Evaluation and comparison of espresso coffees.

The antioxidant profiles of various espresso coffees were established using HPLC with UV-absorbance detection and two rapid, simultaneous, on-line chemical assays that enabled the relative reactivity of sample components to be screened. The assays were based on (i) the colour change associated with reduction of the 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH*); and (ii) the emission of light (chemiluminescence) upon reaction with acidic potassium permanganate. Results from the two approaches were similar and reflected the complex array of antioxidant species present in the samples. However, some differences in selectivity were observed. Chromatograms generated with the chemiluminescence assay contained more peaks, which was ascribed to the greater sensitivity of the reagent towards minor, readily oxidisable sample components. The three coffee samples produced closely related profiles, signifying their fundamentally similar chemical compositions and origin. Nevertheless, the overall intensity and complexity of the samples in both UV absorption and antioxidant assay chromatograms were aligned with the manufacturers description of flavour intensity and character.

Determination of Citrus aurantium protoalkaloids using HPLC with acidic potassium permanganate chemiluminescence detection.

Acidic potassium permanganate chemiluminescence was explored as a sensitive and selective mode of detection for phenolic phenethylamines (adrenergic amines) in consumer products containing Citrus aurantium extracts. Nine commercially available weight-loss products were analysed using rapid reversed-phase chromatography with a monolithic column (separation time of 4 min). The results were in good agreement with package labelling, with some notable exceptions. The products contained a wide concentration range of synephrine and total adrenergic amines, and the difference in consumer intake was even greater when the manufacturers' recommended daily consumption was considered. The quantity of the extract, often specified on the packaging as equivalent grams of dry C. aurantium fruit, was a poor indicator of the concentration of the active ingredients. Methionine, a thioether amino acid contained in some weight-loss products, was identified as a potential interferent for this mode of detection.

Selectivity and potential interference from phenolic compounds in chemiluminescence methods for the determination of synephrine.

Three recently reported chemiluminescence methods (based on reactions with alkaline luminol and hexacyanoferrate(III); acidic cerium(IV) and rhodamine B; and acidic permanganate with polyphosphates) for the determination of synephrine were re-evaluated in terms of their selectivity towards this analyte in comparison to other phenolic compounds. A fourth reagent system, acidic soluble manganese(IV) and formaldehyde, was also examined. Each set of reagents was sensitive towards synephrine (limits of detection were 3 x 10(-9), 5 x 10(-8), 1 x 10(-8) and 1 x 10(-8) mol/L, respectively) but also responded with numerous other phenolic compounds, including some that are present in citrus fruit extracts, dietary supplements and/or biological fluids. It is therefore recommended that the determination of synephrine in these matrices should incorporate physical separation of sample components (e.g. chromatography or electrophoresis). In more general terms, this study illustrates that accurate percentage recoveries for an analyte in spiked samples (without validation against another analytical method) are insufficient to confirm the analytical utility of new flow-injection analysis (FIA) procedures.

Chemiluminescence detection of opium poppy (Papaver somniferum) alkaloids.

A review with 98 references. The determination of the opium poppy (Papaver somniferum) alkaloids and their semi-synthetic derivatives has important applications in industrial process monitoring, clinical analysis and forensic science. Liquid-phase chemiluminescence reagents such as tris(2,2'-bipyridyl)ruthenium(II) and acidic potassium permanganate exhibit remarkable sensitivity and complementary selectivity for many P. somniferum alkaloids, which has been exploited in the development of a range of analytical procedures using flow analysis, high-performance liquid chromatography, capillary electrophoresis and microfluidic instrumentation.

Determination of synephrine in weight-loss products using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection.

Adrenergic amines found in extracts of Citrus aurantium (bitter orange) evoke analytically useful chemiluminescence with acidic potassium permanganate in the presence of polyphosphates. From corrected chemiluminescence spectra, the wavelength of maximum intensity for these reactions was 680+/-5 nm and, using flow injection analysis methodology, limits of detection for synephrine, octopamine, tyramine and hordenine were found to be between 1x10(-9) and 1x10(-8) M. We have applied this method of detection to the rapid determination of synephrine in dietary supplements using monolithic column chromatography.