Lisa Elviri

Nitric Oxide Is Involved in Cadmium-Induced Programmed Cell Death in Arabidopsis Suspension Cultures

Exposure to cadmium (Cd2+) can result in cell death, but the molecular mechanisms of Cd2+ cytotoxicity in plants are not fully understood. Here, we show that Arabidopsis (Arabidopsis thaliana) cell suspension cultures underwent a process of programmed cell death when exposed to 100 and 150 μm CdCl2 and that this process resembled an accelerated senescence, as suggested by the expression of the marker senescence-associated gene12 (SAG12). CdCl2 treatment was accompanied by a rapid increase in nitric oxide (NO) and phytochelatin synthesis, which continued to be high as long as cells remained viable. Hydrogen peroxide production was a later event and preceded the rise of cell death by about 24 h. Inhibition of NO synthesis by NG-monomethyl-arginine monoacetate resulted in partial prevention of hydrogen peroxide increase, SAG12 expression, and mortality, indicating that NO is actually required for Cd2+-induced cell death. NO also modulated the extent of phytochelatin content, and possibly their function, by S-nitrosylation. These results shed light on the signaling events controlling Cd2+ cytotoxicity in plants.

Copper Binding Agents Acting as Copper Ionophores Lead to Caspase Inhibition and Paraptotic Cell Death in Human Cancer Cells

We report a quantitative structure-activity relationship study of a new class of pyrazole-pyridine copper complexes that establishes a clear correlation between the ability to promote copper accumulation and cytotoxicity. Intracellular metal accumulation is maximized when ligand lipophilicity allows the complex to rapidly cross the membrane. Copper and ligand follow different uptake kinetics and reach different intracellular equilibrium concentrations. These results support a model in which the ligand acts as an ionophore for the metal ion, cycling between intra- and extracellular compartments as dissociated or complexed entities. When treating cancer cells with structurally unrelated disulfiram and pyrazole-pyridine copper complexes, as well as with inorganic copper, the same morphological and molecular changes were reproduced, indicating that copper overload is responsible for the cytotoxic effects. Copper-based treatments drive sensitive cancer cells toward paraptotic cell death, a process hallmarked by endoplasmic reticulum stress and massive vacuolization in the absence of apoptotic features. A lack of caspase activation, as observed in copper-treated dying cells, is a consequence of metal-mediated inhibition of caspase-3. Thus, copper acts simultaneously as an endoplasmic reticulum (ER) stress inducer and a caspase-3 inhibitor, forcing the cell into caspase-independent paraptotic death. The establishment of a mechanism of action common to different copper binding agents provides a rationale for the exploitation of copper toxicity as an anticancer tool.

Liquid chromatography-UV determination and liquid chromatography-atmospheric pressure chemical ionization mass spectrometric characterization of sitosterol and stigmasterol in soybean oil

A narrow-bore HPLC-UV method was developed for the analysis of two of the more abundant naturally occurring phytosterols in vegetable oils: sitosterol and stigmasterol. The method enabled detection of the compounds at a concentration of 0.42 microg/ml and quantitation at concentrations of 0.52 and 0.54 microg/ml for sitosterol and stigmasterol, respectively. An excellent linearity was determined over two orders of concentration magnitude (r2 0.999-1.000) and verified by applying the Mandel fitting test (p>0.099) and the lack-of-fit test (p>0.057) performed at the 95% confidence level. A good intra-day precision ranging from 0.15 to 1.16% was calculated at two concentration levels (2 and 100 microg/ml). The inter-day reproducibility was verified on 3 different days by performing an homoscedasticity test and analysis of variance. A solid-phase extraction method was developed on silica cartridges for the isolation of phytosterols from soybean oil providing recovery values of 101+/-9 and 106+/-7% for sitosterol and stigmasterol, respectively. Good accuracy of the method was statistically demonstrated since no matrix effect was found for both the analytes. The developed method was applied to the quantitative assay of phytosterols in a soybean oil sample (61+/-5 mg/100 g of stigmasterol and 118+/-4 mg/100 g sitosterol). The HPLC-atmospheric pressure chemical ionization MS technique enabled the identification of stigmasterol, sitosterol and campesterol in the oil sample.

Spectrophotometric and coulometric detection in the high-performance liquid chromatography of flavonoids and optimization of sample treatment for the determination of quercetin in orange juice.

The capabilities of spectrophotometric and electrochemical detection techniques were investigated for the high-performance liquid chromatographic determination of flavonoids. Liquid chromatographic analyses were performed on eleven compounds belonging to three different classes of flavonoids: flavanone glycosides, flavone and flavonol aglycones. Separation of all compounds examined was carried out under reversed-phase conditions on a C18 narrow-bore column for UV detection, whereas for electrochemical detection, a C18 standard-bore column was used. UV analyses were carried out at 280 nm for flavanones and at 265 nm for flavones and flavonols, whereas controlled-potential coulometric measurements were performed using a porous graphite electrode. Analytical performances of the methods were compared in terms of linearity, limits of detection (LODs) and precision. Linearity over two orders of magnitude and LODs at low-ppm levels (0.06-1 mg/l) were demonstrated for all techniques considered. Instrumental precision in terms of relative standard deviation was found to be between 0 and 5% for the liquid chromatography (LC)-UV system and between 0.6 and 10% for the LC-electrochemical detection (ED) system. The methods developed were applied to the analysis of flavanones and flavonols in a real sample, such as an extract of orange juice. Even though quercetin glycoside is mostly present in orange juice as rutin, other different glycosides of this flavonol could be present; on this basis, the hydrolysis of all glycosides to aglycone allows one to obtain more accurate data on the flavonol concentration in orange juice. To avoid sample degradation and to increase extraction efficiency, quercetin hydrolysis was optimized using a central composite design to investigate the effects of acid concentration and hydrolysis time on extraction recovery.

Multiplexed Determination of Protein Biomarkers Using Metal-Tagged Antibodies and Size Exclusion Chromatography−Inductively Coupled Plasma Mass Spectrometry

A liquid-phase immunoassay was developed for the simultaneous determination of five cancer biomarker proteins: alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), carcinoembryonic antigen (CEA), ovarian tumor antigen (CA125/MUC16), and gastrointestinal tumor antigen (CA19-9). The method was based on the incubation of a serum (or tissue cytosol) with five antibodies, each labeled with a different lanthanide (Pr(3+), Eu(3+), Gd(3+), Ho(3+), and Tb(3+), respectively) followed by the specific determination of the immunocomplex formed by size exclusion chromatography with inductively coupled plasma mass spectrometric detection (SEC-ICPMS). The sensitivity of the method was comparable with that attainable by enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay with the advantages of multiplexed analysis capacity, virtually no sample preparation, and sample amount consumption, ca. 3 times lower than an ELISA test. The method was validated for the analysis of the proteins in human serum and proved to be able to discriminate ovary and uterus tumor tissue samples from those of healthy subjects.

Validation of a liquid chromatography ionspray mass spectrometry method for the analysis of flavanones, flavones and flavonols.

The application of liquid chromatography/mass spectrometry (LC/MS) with a TurboIonspray (TIS) interface was investigated as a new method for the analysis of flavonoids. Eleven compounds belonging to three different classes of flavonoids were studied: eriocitrin, neoeriocitrin, naringin, narirutin, hesperidin, neohesperidin (flavanone glycosides), quercetin, kaempferol, galangin (flavonol aglycones), chrysin, apigenin (flavone aglycones). Chromatographic separations were performed under reversed-phase conditions using a C18 narrow-bore LC column; a mixture of an aqueous solution of formic acid (pH 2.4) and acetonitrile was used as the mobile phase. Isocratic elution was operated in the case of flavanones, whereas gradient elution was used for the simultaneous separation of flavones and flavonols. The adaptability of TIS to high flow applications allows the use of LC eluent flow rates at 200 µL/min without post-column splitting. Qualitative analysis was performed in negative-ion (NI) full-scan mode, whereas response linearity, detection limits and precision of the method were studied under NI selected ion monitoring (SIM) conditions. Characterization of isomers differing in the glycosylation was found to be possible on the basis of different mass spectra. Detection limits in the low-ng range (0.08-0.4 ng) were found, about twenty-fold lower than those reported previously. The method was applied to identify and determine the content of flavonoids in an orange juice sample. Copyright 1999 John Wiley & Sons, Ltd.

Liquid chromatography–electrospray mass spectrometry of β-carotene and xanthophylls: Validation of the analytical method

The investigation of beta-carotene and the xanthophylls beta-cryptoxanthin, lutein, zeaxanthin, canthaxanthin and astaxanthin using reversed-phase liquid chromatography-electrospray mass spectrometry interfaced with TurboIonspray (LC-TurboISP-MS) is described. Two narrow-bore C18 columns connected in series and an isocratic solvent system containing acetonitrile-methanol (0.1 M ammonium acetate)-dichloromethane at a flow-rate of 300 microl/min (without splitting) were used. Operating in the positive-ion mode over m/z 500-650, the effects on the formation of the molecular ion species or adduct ions and the MS detector response were investigated for carotenoids, varying the orifice plate voltage, the ring voltage and the ISP voltage. Both conventional ISP and TurboISP were performed; using the TurboISP-MS system, ionization efficiency increased with respect to ISP-MS, particularly at the highest temperature (500 degrees C). Good results were particularly obtained for beta-carotene, which was detectable at the low ng level, without the use of solution-phase oxidants. Using LC columns and acquiring in single-ion monitoring mode, detection limits were estimated to be in the 0.1-1 ng range; dynamic range was established between one- and two-orders of magnitude. Better sensitivity under positive-ion than negative-ion conditions was demonstrated.

Selective and rapid immunomagnetic bead-based sample treatment for the liquid chromatography-electrospray ion-trap mass spectrometry detection of Ara h3/4 peanut protein in foods

Complex matrices commonly affect the sensitivity and selectivity of liquid chromatography-mass spectrometry (LC-MS) analysis. Thus, selective sample enrichment strategies are useful particularly to analyze organic biomarkers present in low abundance in samples. A selective immunomagnetic extraction procedure to isolate trace peanut allergen protein Ara h3/4 from breakfast cereals combined with microwave-assisted tryptic digestion and liquid chromatography-electrospray ion-trap tandem mass spectrometry (LC-ESI-IT-MS/MS) measurement was developed. Using protein A-coated magnetic bead (MB) support, anti-Ara h3/4 monoclonal antibodies (Abs) were used as selective capture molecules. The results obtained by LC-ESI-IT-MS/MS in terms of limit of detection (3 mg peanuts/kg matrix) and a significantly reduced matrix effect demonstrated that the Ab-coated magnetic bead was very effective to selectively trap Ara h3/4 protein in breakfast cereals. The magnetic bead-based sample treatment followed by LC-IT-MS/MS method here developed can be proposed as very rapid and powerful confirmatory analytical method to verify the reliability of enzyme-linked immunosorbent assay (ELISA) screening methods, since the magnetic bead-LC-IT-MS/MS method combines good sensitivity to the identification capabilities of mass spectrometry.

A voltammetric immunosensor based on nanobiocomposite materials for the determination of alpha-fetoprotein in serum.

The development of a voltammetric immunosensor for determination of alpha-fetoprotein (AFP) in serum is presented. ELISA assays with voltammetric reading were carried out exploiting the peculiar properties of nanobiocomposite materials based on gold nanoparticles for the immobilization of Antibody (Ab)/Antigen/Antibody-HRP (Horseradish Peroxidase) sandwich on the glassy carbon (GC) electrode surface. The electrochemical transduction was mediated by thionin, which was used in its monomeric form dissolved in the reading solution, so avoiding critical immobilization procedures. The study was aimed at the development and validation of an immunosensor able to provide results in short time, simple to use, rugged and cost-effective for AFP monitoring purposes. A crucial aspect of the study was the development of an experimental protocol leading to highly standardized and consequently reproducible sensors. Two-way analysis of variance (ANOVA) was applied to study the effect of the concentration of the solutions used for the incubation of the antibodies. The sensor was validated in serum assessing stability of the immunocomplex, linearity of response, limit of detection (3.7 ng/ml) and limit of quantitation (11 ng/ml), precision (intra- and inter-sensor repeatability) and recovery rate (103%). The stability of the GC/Ab functionalized substrate was demonstrated over one month, showing variation coefficients below 5%. Experiments carried out with real samples of clinical interest evidenced that the developed immunosensor can be considered as powerful tool in cancer screening programmes.

Particle-packed column versus silica-based monolithic column for liquid chromatography-electrospray-linear ion trap-tandem mass spectrometry multiallergen trace analysis in foods.

A bicarbonate buffer-based extraction method for the simultaneous analysis of five nut allergens (Ana o 2, cashew-nut; Cor a 9, hazelnut; Pru 1, almond; Ara h3/4, peanut; Jug r 4, walnut) in cereals and biscuits using liquid chromatography-electrospray-linear ion trap-tandem mass spectrometry (LC-ESI-LIT-MS(2)) was developed and validated. The method was based on our earlier published LC-MS(2)-based method in a research frame aimed at the identification and determination of hidden allergens in foods by using selective biomarker peptides. A C18 particle-packed column and a silica-based C18 monolithic column were compared in terms of chromatographic performances, such as peak shape, resolution, analysis time and selectivity. The C18 particle-packed column exhibited better performances and was further used for method development and validation. By operating under MS(2) selected reaction monitoring (SRM) acquisition mode, linearity, limits of detection (LOD) and quantitation, trueness and precision were evaluated on breakfast samples enriched with a mix of the five nuts. Good linearity of the matrix matched-calibration curves was obtained and detection limit values generally varied from 14 to 55 mg nut/kg matrix. Recoveries were in the 76±4% to 94±3% range with RSD <15%. The capabilities of LIT to perform MS(n) fragmentation was exploited to improve selectivity of the analysis, and the LC-(SRM) MS(2) method was compared in terms of LOD, linearity, precision and accuracy with a LC-(SRM) MS(3) method. Finally, both the LC-MS(2) and LC-MS(3) methods were successfully applied to the analysis of nut traces in commercially available breakfast cereals and biscuits.