Investigation of urinary volatile organic metabolites as potential cancer biomarkers by solid-phase microextraction in combination with gas chromatography-mass spectrometry

BACKGROUND: Non-invasive diagnostic strategies aimed at identifying biomarkers of cancer are of great interest for early cancer detection. Urine is potentially a rich source of volatile organic metabolites (VOMs) that can be used as potential cancer biomarkers. Our aim was to develop a generally reliable, rapid, sensitive, and robust analytical method for screening large numbers of urine samples, resulting in a broad spectrum of native VOMs, as a tool to evaluate the potential of these metabolites in the early diagnosis of cancer. METHODS: To investigate urinary volatile metabolites as potential cancer biomarkers, urine samples from 33 cancer patients (oncological group: 14 leukaemia, 12 colorectal and 7 lymphoma) and 21 healthy (control group, cancer-free) individuals were qualitatively and quantitatively analysed. Dynamic solid-phase microextraction in headspace mode (dHS-SPME) using a carboxenpolydimethylsiloxane (CAR/PDMS) sorbent in combination with GC-qMS-based metabolomics was applied to isolate and identify the volatile metabolites. This method provides a potential non-invasive method for early cancer diagnosis as a first approach. To fulfil this objective, three important dHS-SPME experimental parameters that influence extraction efficiency (fibre coating, extraction time and temperature of sampling) were optimised using a univariate optimisation design. The highest extraction efficiency was obtained when sampling was performed at 501C for 60min using samples with high ionic strengths (17% sodium chloride, wv 1) and under agitation. RESULTS: A total of 82 volatile metabolites belonging to distinct chemical classes were identified in the control and oncological groups. Benzene derivatives, terpenoids and phenols were the most common classes for the oncological group, whereas ketones and sulphur compounds were the main classes that were isolated from the urine headspace of healthy subjects. The results demonstrate that compound concentrations were dramatically different between cancer patients and healthy volunteers. The positive rates of 16 patients among the 82 identified were found to be statistically different (Po0.05). A significant increase in the peak area of 2-methyl3-phenyl-2-propenal, p-cymene, anisole, 4-methyl-phenol and 1,2-dihydro-1,1,6-trimethyl-naphthalene in cancer patients was observed. On average, statistically significant lower abundances of dimethyl disulphide were found in cancer patients. CONCLUSIONS: Gas chromatographic peak areas were submitted to multivariate analysis (principal component analysis and supervised linear discriminant analysis) to visualise clusters within cases and to detect the volatile metabolites that are able to differentiate cancer patients from healthy individuals. Very good discrimination within cancer groups and between cancer and control groups was achieved.

Effectiveness of different solid-phase microextraction fibres for differentiation of selected Madeira island fruits based on their volatile metabolite profile: identification of novel compounds

A headspace solid-phase microextraction (HS-SPME) procedure based on five commercialised fibres (85 μm polyacrylate – PA, 100 μm polydimethylsiloxane – PDMS, 65 μm polydimethylsiloxane/divinylbenzene – PDMS/DVB, 70 μm carbowax/divinylbenzene – CW/DVB and 85 μm carboxen/polydimethylsiloxane – CAR/PDMS) is presented for the characterization of the volatile metabolite profile of four selected Madeira island fruit species, lemon (Citrus limon), kiwi (Actinidia deliciosa), papaya (Carica papaya L.) and Chickasaw plum (Prunus angustifolia). The isolation of metabolites was followed by thermal desorption gas chromatography–quadrupole mass spectrometry (GC–qMS) methodology. The performance of the target fibres was evaluated and compared. The SPME fibre coated with CW/DVB afforded the highest extraction efficiency in kiwi and papaya pulps, while in lemon and plum the same was achieved with PMDS/DVB fibre. This procedure allowed for the identification of 80 compounds, 41 in kiwi, 24 in plums, 23 in papaya and 20 in lemon. Considering the best extraction conditions, the most abundant volatiles identified in kiwi were the intense aldehydes and ethyl esters such as (E)-2-hexenal and ethyl butyrate, while in Chicasaw plum predominate 2-hexenal, 2-methyl-4-pentenal, hexanal, (Z)-3-hexenol and cyclohexylene oxide. The major compounds identified in the papaya pulp were benzyl isothiocyanate, linalool oxide, furfural, hydroxypropanone, linalool and acetic acid. Finally, lemon was shown to be the most divergent of the four fruits, being its aroma profile composed almost exclusively by terpens, namely limonene, γ-terpinene, o-cymene and α-terpinolene. Thirty two volatiles were identified for the first time in the fruit or close related species analysed and 14 volatiles are reported as novel volatile metabolites in fruits. This includes 5 new compounds in kiwi (2-cyclohexene-1,4-dione, furyl hydroxymethyl ketone, 4-hydroxydihydro-2(3H)-furanone, 5-acetoxymethyl-2-furaldehyde and ethanedioic acid), 4 in plum (4-hydroxydihydro-2(3H)-furanone, 5-methyl-2-pyrazinylmethanol, cyclohexylene oxide and 1-methylcyclohexene), 4 in papaya (octaethyleneglycol, 1,2-cyclopentanedione, 3-methyl-1,2-cyclopentanedione and 2-furyl methyl ketone) and 2 in lemon (geranyl farnesate and safranal). It is noteworthy that among the 15 volatile metabolites identified in papaya, 3-methyl-1,2-cyclopentanedione was previously described as a novel PPARγ (peroxisome proliferator-activated receptor γ) agonist, having a potential to minimize inflammation.

A new and improved strategy combining a dispersive-solid phase extraction-based multiclass method with ultra high pressure liquid chromatography for analysis of low molecular weight polyphenols in vegetables

This paper reports on the development and optimization of a modified Quick, Easy, Cheap Effective, Rugged and Safe (QuEChERS) based extraction technique coupled with a clean-up dispersive-solid phase extraction (dSPE) as a new, reliable and powerful strategy to enhance the extraction efficiency of free low molecular-weight polyphenols in selected species of dietary vegetables. The process involves two simple steps. First, the homogenized samples are extracted and partitioned using an organic solvent and salt solution. Then, the supernatant is further extracted and cleaned using a dSPE technique. Final clear extracts of vegetables were concentrated under vacuum to near dryness and taken up into initial mobile phase (0.1% formic acid and 20% methanol). The separation and quantification of free low molecular weight polyphenols from the vegetable extracts was achieved by ultrahigh pressure liquid chromatography (UHPLC) equipped with a phodiode array (PDA) detection system and a Trifunctional High Strength Silica capillary analytical column (HSS T3), specially designed for polar compounds. The performance of the method was assessed by studying the selectivity, linear dynamic range, the limit of detection (LOD) and limit of quantification (LOQ), precision, trueness, and matrix effects. The validation parameters of the method showed satisfactory figures of merit. Good linearity (View the MathML sourceRvalues2>0.954; (+)-catechin in carrot samples) was achieved at the studied concentration range. Reproducibility was better than 3%. Consistent recoveries of polyphenols ranging from 78.4 to 99.9% were observed when all target vegetable samples were spiked at two concentration levels, with relative standard deviations (RSDs, n = 5) lower than 2.9%. The LODs and the LOQs ranged from 0.005 μg mL−1 (trans-resveratrol, carrot) to 0.62 μg mL−1 (syringic acid, garlic) and from 0.016 μg mL−1 (trans-resveratrol, carrot) to 0.87 μg mL−1 ((+)-catechin, carrot) depending on the compound. The method was applied for studying the occurrence of free low molecular weight polyphenols in eight selected dietary vegetables (broccoli, tomato, carrot, garlic, onion, red pepper, green pepper and beetroot), providing a valuable and promising tool for food quality evaluation.

In-depth search focused on furans, lactones, volatile phenols, and acetals as potential age markers of Madeira wines by comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry combined with solid phase microextraction

The establishment of potential age markers of Madeira wine is of paramount significance as it may contribute to detect frauds and to ensure the authenticity of wine. Considering the chemical groups of furans, lactones, volatile phenols, and acetals, 103 volatile compounds were tentatively identified; among these, 71 have been reported for the first time in Madeira wines. The chemical groups that could be used as potential age markers were predominantly acetals, namely, diethoxymethane, 1,1-diethoxyethane, 1,1-diethoxy-2-methyl-propane, 1-(1-ethoxyethoxy)-pentane, trans-dioxane and 2-propyl-1,3-dioxolane, and from the other chemical groups, 5-methylfurfural and cis-oak-lactone, independently of the variety and the type of wine. GC × GC-ToFMS system offers a more useful approach to identify these compounds compared to previous studies using GC−qMS, due to the orthogonal systems, that reduce coelution, increase peak capacity and mass selectivity, contributing to the establishment of new potential Madeira wine age markers. Remarkable results were also obtained in terms of compound identification based on the organized structure of the peaks of structurally related compounds in the GC × GC peak apex plots. This information represents a valuable approach for future studies, as the ordered-structure principle can considerably help the establishment of the composition of samples. This new approach provides data that can be extended to determine age markers of other types of wines.

Optimisation of solid-phase microextraction combined with gas chromatography–mass spectrometry based methodology to establish the global volatile signature in pulp and skin of Vitis vinifera L. grape varieties

The volatiles (VOCs) and semi-volatile organic compounds (SVOCs) responsible for aroma are mainly present in skin of grape varieties. Thus, the present investigation is directed towards the optimisation of a solvent free methodology based on headspace-solid-phase microextraction (HS-SPME) combined with gas chromatography–quadrupole mass spectrometry (GC–qMS) in order to establish the global volatile composition in pulp and skin of Bual and Bastardo Vitis vinifera L. varieties. A deep study on the extraction-influencing parameters was performed, and the best results, expressed as GC peak area, number of identified compounds and reproducibility, were obtained using 4 g of sample homogenised in 5 mL of ultra-pure Milli-Q water in a 20 mL glass vial with addition of 2 g of sodium chloride (NaCl). A divinylbenzene/carboxen/polydimethylsiloxane fibre was selected for extraction at 60 °C for 45 min under continuous stirring at 800 rpm. More than 100 VOCs and SVOCs, including 27 monoterpenoids, 27 sesquiterpenoids, 21 carbonyl compounds, 17 alcohols (from which 2 aromatics), 10 C13 norisoprenoids and 5 acids were identified. The results showed that, for both grape varieties, the levels and number of volatiles in skin were considerably higher than those observed in pulp. According to the data obtained by principal component analysis (PCA), the establishment of the global volatile signature of grape and the relationship between different part of grapes—pulp and skin, may be an useful tool to winemaker decision to define the vinification procedures that improves the organoleptic characteristics of the corresponding wines and consequently contributed to an economic valorization and consumer acceptance.

New developmental stages for common marmosets (Callithrix jacchus) using mass and age variables obtained by K-means algorithm and self-organizing maps (SOM)

LEÃO, Adriano de Castro; DÓRIA NETO, Adrião Duarte; SOUSA, Maria Bernardete Cordeiro de. New developmental stages for common marmosets (Callithrix jacchus) using mass and age variables obtained by K-means algorithm and self-organizing maps (SOM). Computers in Biology and Medicine, v. 39, p. 853-859, 2009

Observer-based state-feedback versus H-infinity output feedback control solved by LMI approach for applications in smart structures

This paper aims with the use of linear matrix inequalities approach (LMIs) for application in active vibration control problems in smart strutures. A robust controller for active damping in a panel was designed with piezoelectrical actuators in optimal locations for illustration of the main proposal. It was considered, in the simulations of the closed-loop, a model identified by eigensystem realization algorithm (ERA) and reduced by modal decomposition. We tested two differents techniques to solve the problem. The first one uses LMI approach by state-feedback based in an observer design, considering several simultaneous constraints as: a decay rate, limited input on the actuators, bounded output peak (output energy) and robustness to parametic uncertainties. The results demonstrated the vibration attenuation in the structure by controlling only the first modes and the increased damping in the bandwidth of interest. However, it is possible to occur spillover effects, because the design has not been done considering the dynamic uncertainties related with high frequencies modes. In this sense, the second technique uses the classical H. output feedback control, also solved by LMI approach, considering robustness to residual dynamic to overcome the problem found in the first test. The results are compared and discussed. The responses shown the robust performance of the system and the good reduction of the vibration level, without increase mass.

Numeric simulation of pollutant dispersion by a control-volume based on finite element method

The dispersion of pollutants in the environment is an issue of great interest as it directly affects air quality, mainly in large cities. Experimental and numerical tools have been used to predict the behavior of pollutant species dispersion in the atmosphere. A software has been developed based on the control-volume based on the finite element method in order to obtain two-dimensional simulations of Navier-Stokes equations and heat or mass transportation in regions with obstacles, varying position of the pollutant source. Numeric results of some applications were obtained and, whenever possible, compared with literature results showing satisfactory accordance. Copyright (C) 2010 John Wiley & Sons, Ltd.

Freshwater stingrays: study of epidemiologic, clinic and therapeutic aspects based on 84 envenomings in humans and some enzymatic activities of the venom

Freshwater stingrays are very common in the Parana, Paraguay, Araguaia, and Tocantins Rivers and tributaries in Brazil. This study presents the clinical aspects of 84 patients injured by freshwater stingrays. Intense pain was the most conspicuous symptom. Skin necrosis was observed in a high percentage of the victims, mostly fishermen and bathers. The initial therapeutic procedures, like immersion of the affected member in hot water were effective in the initial phases of the envenoming, especially in the control of the acute pain; however, they did not prevent skin necrosis. By SDS-PAGE, the freshwater stingray (Potamotrygon falkneri) venom extract presented a major band of approximately 12 kDa. Several other components distributed between 15 and 130 kDa were detected in the venom extract. Many components with molecular mass above 80 and 100 kDa have gelatinolytic and caseinolytic activities, respectively. Hyaluronidase activity was detected only in a component around 84 kDa in P. falkneri venom extract. Our results demonstrated that the presence of these enzymes could explain partially the local clinical pictures presented by patients wounded by freshwater stingray. (C) 2004 Elsevier Ltd. All rights reserved.

Traumatic subdural hygromas: Proposed pathogenesis based classification

Background: Traumatic subdural hygroma (TSHy) is an accumulation of cerebrospinal fluid (CSF) in the subdural space after head injury. It appears to be relatively common, but its onset time and natural history are not well defined. Considered a benign epiphenomenon of trauma, the pathogenesis of TSHy is still unclear and many questions remain unanswered. This study adds to the information on TSHy, and proposes a classification based on pathogenesis.Methods: Thirty-four consecutive adult patients with TSHy were analyzed for clinical evolution and serial CT scan, during a period of several months. TSHy diagnosis was based on published CT scan criteria of hypodense subdural collection after trauma, without enhancement and neomembrane, with a minimum distance of 3 mm between the skull and brain. Ventricle size was analyzed by calculating the bicaudate index (BCI). For comparison, the BCI was measured from CT scan at three moments: admission, at time of TSHy diagnosis, and from last CT scan.Results: There were 34 patients, aged between 16 and 85 years (mean 40), half of them were below 40 years. Road traffic crashes were the main cause of head injury. The mean time for hygroma diagnosis was 9 days. Twenty-one patients (61.8%) underwent conservative treatment for TSHy and 13 (38.2%), surgical treatment. TSHy are early lesions and can be detected in the first 24 hours after trauma, usually as small subdural effusion (SSEff). Based on clinical and CT scan findings, we divided the 34 patients into 3 groups, (Ia and Ib) without evident mass effect and (II) with evident mass effect. Group Ia includes patients without ventricle dilation; Ib, patients with associated ventricle dilations.Conclusions: SSEff detected in the first 24 hours posttrauma in our series evolved into TSHy suggesting that this is an early lesion; all THSy were divided in three groups according to the pathophysiologic mechanism. These three groups probably represent a continuum of CSF absorption impairment. Group la represents what most authors consider a simple hygroma, with no impairment on CSF absorption. Group Ib represent the external hydrocephalus form with various degrees of CSF imbalance, and group II were the cases presenting marked mass effect.

Global gene expression of the inner cell mass and trophectoderm of the bovine blastocyst

Background: The first distinct differentiation event in mammals occurs at the blastocyst stage when totipotent blastomeres differentiate into either pluripotent inner cell mass (ICM) or multipotent trophectoderm (TE). Here we determined, for the first time, global gene expression patterns in the ICM and TE isolated from bovine blastocysts. The ICM and TE were isolated from blastocysts harvested at day 8 after insemination by magnetic activated cell sorting, and cDNA sequenced using the SOLiD 4.0 system.Results: A total of 870 genes were differentially expressed between ICM and TE. Several genes characteristic of ICM (for example, NANOG, SOX2, and STAT3) and TE (ELF5, GATA3, and KRT18) in mouse and human showed similar patterns in bovine. Other genes, however, showed differences in expression between ICM and TE that deviates from the expected based on mouse and human.Conclusion: Analysis of gene expression indicated that differentiation of blastomeres of the morula-stage embryo into the ICM and TE of the blastocyst is accompanied by differences between the two cell lineages in expression of genes controlling metabolic processes, endocytosis, hatching from the zona pellucida, paracrine and endocrine signaling with the mother, and genes supporting the changes in cellular architecture, stemness, and hematopoiesis necessary for development of the trophoblast.

New analytical procedure based on a cellulose bag and ionic exchanger with p-aminobenzoic acid groups for differentiation of labile and inert metal species in aquatic systems

A new procedure was developed for the in situ characterization of the lability of metal species in aquatic systems by using a system equipped with a diffusion membrane and cellulose organomodified with p-aminobenzoic acid groups (DM-Cell-PAB). To this end, the DM-Cell-PAB system was prepared by adding cellulose organomodified with p-aminobenzoic acid groups (Cell-PAB) to pre-purified cellulose bags. After the DM-Cell-PAB system was sealed, it was examined in the laboratory to evaluate the influence of complexation time, mass of exchanger, pH, metal ions (Cu, Cd, Fe, Mn, and Ni), and concentration of organic matter on the relative lability of metal species. It was found that the pH and kinetics strongly influence the process of metal complexation by the DM-Cell-PAB system. At all pH levels, Cd, Mn, and Ni showed lower complexation with Cell-PAB resin than Cu and Fe metals. Note that relative lability of metals complexed to aquatic humic substances (AHS) in the presence of Cell-PAB resin showed the following order: Cu congruent to Fe >> Ni > Mn=Cd. The results presented here also indicate that increasing the AHS concentration decreases the lability of metal species by shifting the equilibrium to AHS-metal complexes. Our results indicate that the system under study offers an interesting alternative that can be applied to in situ experiments for differentiation of labile and inert metal species in aquatic systems.

The shielding effect of glycerol against protein ionization in electrospray mass spectrometry

Most commercial recombinant proteins used as molecular biology tools, as well as many academically made preparations, are generally maintained in the presence of high glycerol concentrations after purification to maintain their biological activity. The present study shows that larger proteins containing high concentrations of glycerol are not amenable to analysis using conventional electrospray ionization mass spectrometry (ESI-MS) interfaces. In this investigation the presence of 25% (v/v) glycerol suppressed the signals of Taq DNA polymerase molecules, while 1% (v/v) glycerol suppressed the signal of horse heart myoglobin. The signal suppression was probably caused by the interaction of glycerol molecules with the proteins to create a shielding effect that prevents the ionization of the basic and/or acidic groups in the amino acid side chains. To overcome this difficulty the glycerol concentration was decreased to 5% (v/v) by dialyzing the Taq polymerase solution against water, and the cone voltage in the ESI triple-quadrupole mass spectrometer was set at 80-130 V. This permitted observation of a mass spectrum that contained ions corresponding to protonation of up to 50% of the ionizable basic groups. In the absence of glycerol up to 85% of the basic groups of Taq polymerase became ionized, as observed in the mass spectrum at relatively low cone voltages. An explanation of these and other observations is proposed, based on strong interactions between the protein molecules and glycerol. For purposes of comparison similar experiments were performed on myoglobin, a small protein with 21 basic groups, whose ionization was apparently suppressed in the presence of 1% (v/v) glycerol, since no mass spectrum could be obtained even at high cone voltages. Copyright (C) 2003 John Wiley Sons, Ltd.

A mass-transportation approach to a one dimensional fluid mechanics model with nonlocal velocity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Measurement of the top quark mass in the lepton plus jets channel using the ideogram method

A measurement of the top quark mass using events with one charged lepton, missing transverse energy, and jets in the final state, collected by the D0 detector from p (p) over bar collisions at root s=1.96 TeV at the Fermilab Tevatron collider, is presented. A constrained fit is used to fully reconstruct the kinematics of the events. For every event a top quark mass likelihood is calculated taking into account all possible jet assignments and the probability that an event is signal or background. Lifetime-based identification of b jets is employed to enhance the separation between t (t) over bar signal and background from other physics processes and to improve the assignment of the observed jets to the quarks in the t (1) over bar hypothesis. We extract a multiplicative jet energy scale (JES) factor in situ, greatly reducing the systematic effect related to the jet energy measurement. In a data sample with an integrated luminosity of 425 pb(-1), we observe 230 candidate events, with an estimated background of 123 events, and measure m(t)=173.7 +/- 4.4(stat+JES)(-2.0)(+2.1)(syst) GeV. This result represents the first application of the ideogram technique to the measurement of the top quark mass in lepton+jets events.