Maria Francesca Riccio

Baseline resolution of isomers by traveling wave ion mobility mass spectrometry: investigating the effects of polarizable drift gases and ionic charge distribution

We have studied the behavior of isomers and analogues by traveling wave ion mobility mass spectrometry (TWIM-MS) using drift-gases with varying masses and polarizabilities. Despite the reduced length of the cell (18 cm), a pair of constitutional isomers, N-butylaniline and para-butylaniline, with theoretical collision cross-section values in helium (ΩHe ) differing by as little as 1.2 Å(2) (1.5%) but possessing contrasting charge distribution, showed baseline peak-to-peak resolution (Rp-p ) for their protonated molecules, using carbon dioxide (CO2), nitrous oxide (N2O) and ethene (C2H4 ) as the TWIM drift-gas. Near baseline Rp-p was also obtained in CO2 for a group of protonated haloanilines (para-chloroaniline, para-bromoaniline and para-iodoaniline) which display contrasting masses and theoretical ΩHe , which differ by as much as 15.7 Å(2) (19.5%) but similar charge distributions. The deprotonated isomeric pair of trans-oleic acid and cis-oleic acid possessing nearly identical theoretical ΩHe and ΩN2 as well as similar charge distributions, remained unresolved. Interestingly, an inversion of drift-times were observed for the 1,3-dialkylimidazolium ions when comparing He, N2 and N2O. Using density functional theory as a means of examining the ions electronic structure, and He and N2-based trajectory method algorithm, we discuss the effect of the long-range charge induced dipole attractive and short-range Van der Waals forces involved in the TWIM separation in drift-gases of differing polarizabilities. We therefore propose that examining the electronic structure of the ions under investigation may potentially indicate whether the use of more polarizable drift-gases could improve separation and the overall success of TWIM-MS analysis.

Grape juice concentrate prevents oxidative DNA damage in peripheral blood cells of rats subjected to a high-cholesterol diet

The goal of the present study was to investigate whether subchronic treatment with grape juice concentrate is able to protect liver and peripheral blood cells against cholesterol-induced injury in rats. The effects of the grape juice concentrate treatment on histopathological changes, immunohistochemistry for cyclo-oxygenase-2 (COX-2), and basal and oxidative DNA damage induced by H2O2 using a single-cell gel (comet) assay were evaluated. Male Wistar rats (n 18) were divided into three groups: group 1--negative control; group 2--cholesterol at 1 % (w/w) in their diet, treated for 5 weeks; group 3--cholesterol at 1 % in their chow, treated for 5 weeks, and grape juice concentrate at 222 mg/d in their drinking-water in the final week only. The results indicated that the treatment with grape juice concentrate did not show remarkable differences regarding liver tissue in group 3 compared with group 2. However, grape juice concentrate was able to decrease oxidative DNA damage induced by H2O2 in peripheral blood cells, as depicted by the tail moment results. COX-2 expression in the liver did not show statistically significant differences (P>0·05) between groups. Taken together, the present results suggest that the administration of subchronic grape juice concentrate prevents oxidative DNA damage in peripheral blood cells.

Easy Ambient Sonic-Spray Ionization Mass Spectrometric of Olive Oils: Quality Control and Certification of Geographical Origin

Herein, we show that easy ambient sonic spray ionization mass spectrometry in the negative ion mode [EASI(-)-MS] of water:methanol extracts of olive oil samples from 5 different countries (Portugal, Italy, Spain, Lebanon, and Greece) provides very characteristic profiles of chemotaxonomic markers, that is, free fatty acids and phenols. These EASI(-)-MS fingerprints, acquired with great speed and simplicity after minimal sample preparation, permits secure identification of the samples as olive oils via their unique profiles of fatty acids plus phenolic constituents as well the certification of geographical regions via characteristic features of the profiles of phenolic constituents.

Mass spectrometry fingerprinting of media used for in vitro production of bovine embryos

Using the bovine species as a biological model, direct infusion chip-based nano-electrospray ionization mass spectrometry (nano-ESI-MS) fingerprinting in the positive ion mode is used to obtain fast chemical profiles of media used for in vitro production of bovine embryos. Nano-ESI-MS fingerprinting is useful for characterization and routine quality control requiring no sample pre-separation, being able to differentiate four different media (IVM, IVF, SOF and HSOF) via principal component analysis (PCA). For media stored at +4 degrees C for up to 45 days, no significant (p>0.05) variation was observed in cleavage and blastocyst rate development, as well as in the nano-ESI-MS chemical profiles. For media exposed to a heat shock (60 degrees C for 3 h), no significant decrease (p>0.05) in embryo development rates was observed, but nano-ESI-MS profiles were quite distant from fresh control media in the PCA. For frozen media (-70 degrees C for 2 months), again no significant variation (p>0.05) in embryo development was noticed, but nano-ESI-MS profiles from all media were significantly affected. These results indicate that nano-ESI(+)-MS fingerprinting was able to characterize different media based on their specific chemical profile. The technique seems therefore applicable as a routine quality control assay, detecting, for example, compositional changes after temperature variations that may affect post-transfer embryo viability.

19-base pair deletion polymorphism of the dihydrofolate reductase (DHFR) gene: maternal risk of Down syndrome and folate metabolism

CONTEXT AND OBJECTIVE Polymorphisms in genes involved in folate metabolism may modulate the maternal risk of Down syndrome (DS). This study evaluated the influence of a 19-base pair (bp) deletion polymorphism in intron-1 of the dihydrofolate reductase (DHFR) gene on the maternal risk of DS, and investigated the association between this polymorphism and variations in the concentrations of serum folate and plasma homocysteine (Hcy) and plasma methylmalonic acid (MMA). DESIGN AND SETTING Analytical cross-sectional study carried out at Faculdade de Medicina de São José do Rio Preto (Famerp). METHODS 105 mothers of individuals with free trisomy of chromosome 21, and 184 control mothers were evaluated. Molecular analysis on the polymorphism was performed using the polymerase chain reaction (PCR) through differences in the sizes of fragments. Folate was quantified by means of chemiluminescence, and Hcy and MMA by means of liquid chromatography and sequential mass spectrometry. RESULTS There was no difference between the groups in relation to allele and genotype frequencies (P = 0.44; P = 0.69, respectively). The folate, Hcy and MMA concentrations did not differ significantly between the groups, in relation to genotypes (P > 0.05). CONCLUSIONS The 19-bp deletion polymorphism of DHFR gene was not a maternal risk factor for DS and was not related to variations in the concentrations of serum folate and plasma Hcy and MMA in the study population.

DHFR 19-bp Deletion and SHMT C1420T Polymorphisms and Metabolite Concentrations of the Folate Pathway in Individuals with Down Syndrome

BACKGROUND Down syndrome (DS) results from the presence and expression of three copies of the genes located on chromosome 21. Studies have shown that, in addition to overexpression of the Cystathionine β-synthase (CBS) gene, polymorphisms in genes involved in folate/homocysteine (Hcy) metabolism may also influence the concentrations of metabolites of this pathway. AIM Investigate the association between Dihydrofolate reductase (DHFR) 19-base pair (bp) deletion and Serine hydroxymethyltransferase (SHMT) C1420T polymorphisms and serum folate and plasma Hcy and methylmalonic acid (MMA) concentrations in 85 individuals with DS. METHODS Molecular analysis of the DHFR 19-bp deletion and SHMT C1420T polymorphisms was performed by polymerase chain reaction (PCR) by difference in the size of fragments and real-time PCR allelic discrimination, respectively. Serum folate was quantified by chemiluminescence and plasma Hcy and MMA by liquid chromatography-tandem mass spectrometry. RESULTS Individuals with DHFR DD/SHMT TT genotypes presented increased folate concentrations (p=0.004) and the DHFR II/SHMT TT genotypes were associated with increased MMA concentrations (p=0.008). In addition, the MMA concentrations were negatively associated with age (p=0.04). CONCLUSION There is an association between DHFR DD/SHMT TT and DHFR II/SHMT TT combined genotypes and folate and MMA concentrations in individuals with DS.

Visualizing inhibition of fatty acid synthase through mass spectrometric analysis of mitochondria from melanoma cells

Fatty acid synthase (FASN) is the metabolic enzyme responsible for the endogenous synthesis of the saturated long-chain fatty acid palmitate. In contrast to most normal cells, FASN is overexpressed in a variety of human cancers including cutaneous melanoma, in which its levels of expression are associated with a poor prognosis and depth of invasion. Recently, we have demonstrated the mitochondrial involvement in FASN inhibition-induced apoptosis in melanoma cells. Herein we compare, via electrospray ionization mass spectrometry (ESI-MS), free fatty acids (FFA) composition of mitochondria isolated from control (EtOH-treated cells) and Orlistat-treated B16-F10 mouse melanoma cells. Principal component analysis (PCA) was applied to the ESI-MS data and found to separate the two groups of samples. Mitochondria from control cells showed predominance of six ions, that is, those of m/z 157 (Pelargonic, 9:0), 255 (Palmitic, 16:0), 281 (Oleic, 18:1), 311 (Arachidic, 20:0), 327 (Docosahexaenoic, 22:6) and 339 (Behenic, 22:0). In contrast, FASN inhibition with Orlistat changes significantly mitochondrial FFA composition by reducing synthesis of palmitic acid, and its elongation and unsaturation products, such as arachidic and behenic acids, and oleic acid, respectively. ESI-MS of mitochondria isolated from Orlistat-treated cells presented therefore three major ions of m/z 157 (Pelargonic, 9:0), 193 (unknown) and 199 (Lauric, 12:0). These findings demonstrate therefore that FASN inhibition by Orlistat induces significant changes in the FFA composition of mitochondria.