CE-ESI-MS metabolic fingerprinting of Leishmania resistance to antimony treatment

Metabolomics has become an invaluable tool to unveil biology of pathogens, with immediate application to chemotherapy. It is currently accepted that there is not one single technique capable of obtaining the whole metabolic fingerprint of a biological system either due to their different physical-chemical properties or concentrations. In this work, we have explored the capability of capillary electrophoresis mass spectrometry with a sheathless interface with electrospray ionization (CE-ESI-TOF-MS) to separate metabolites in order to be used as a complementary technique to LC. As proof of concept, we have compared the metabolome of Leishmania infantum promastigotes BCN 150 (Sb (III) IC50 = 20.9 mu M) and its variation when treated with 120 mu M of Sb(III) potassium tartrate for 12 h, as well as with its Sb(III) resistant counterpart obtained by growth of the parasites under increasing Sb(III) in a step-wise manner up to 180 mu M. The number of metabolites compared were of 264 for BCN150 Sb(III) treated versus nontreated and of 195 for Sb(III) resistant versus susceptible parasites. After successive data filtering, differences in seven metabolites identified in databases for Leishmania pathways, showed the highest significant differences, corresponding mainly to amino acids or their metabolite surrogates. Most of them were assigned to sulfur containing amino acids and polyamine biosynthetic pathways, of special relevance considering the deterioration of the thiol-dependent redox metabolism in Leishmania by Sb(III). Given the low concentrations typical for most of these metabolites, the assay can be considered a success that should be explored for new biological questions.

Solid-phase microextraction combined with comprehensive two-dimensional gas chromatography for fatty acid profiling of cell wall phospholipids

The combination of solid-phase microextraction (SPME) with comprehensive two-dimensional gas chromatography is evaluated here for fatty acid (FA) profiling of the glycerophospholipid fraction from human buccal mucosal cells. A base-catalyzed derivatization reaction selective for polar lipids such as glycerophospholipid was adopted. SPME is compared to a miniaturized liquidliquid extraction procedure for the isolation of FA methyl esters produced in the derivatization step. The limits of detection and limits of quantitation were calculated for each sample preparation method. Because of its lower values of limits of detection and quantitation, SPME was adopted. The extracted analytes were separated, detected, and quantified by comprehensive two-dimensional gas chromatography with flame ionization detection (FID). The combination of SPME and comprehensive two-dimensional gas chromatography with FID, using a selective derivatization reaction in the preliminary steps, proved to be a simple and fast procedure for FA profiling, and was successfully applied to the analysis of adult human buccal mucosal cells.

Characterization of an extrapolation chamber for low-energy X-rays: Experimental and Monte Carlo preliminary results

The extrapolation chamber is a parallel-plate ionization chamber that allows variation of its air-cavity volume. In this work, an experimental study and MCNP-4C Monte Carlo code simulations of an ionization chamber designed and constructed at the Calibration Laboratory at IFEN to be used as a secondary dosimetry standard for low-energy X-rays are reported. The results obtained were within the international recommendations, and the simulations showed that the components of the extrapolation chamber may influence its response up to 11.0%. (C) 2011 Elsevier Ltd. All rights reserved.

Enantioselective analysis of unbound tramadol, O-desmethyltramadol and N-desmethyltramadol in plasma by ultrafiltration and LC-MS/MS: Application to clinical pharmacokinetics

This study describes the enantioselective analysis of unbound and total concentrations of tramadol and its main metabolites O-desmethyltramadol (M1) and N-desmethyltramadol (M2) in human plasma. Sample preparation was preceded by an ultrafiltration step to separate the unbound drug. Both the ultrafiltrate and plasma samples were submitted to liquid/liquid extraction with methyl t-butyl ether. Separation was performed on a Chiralpak (R) AD column and tandem mass spectrometry consisting of an electrospray ionization source, positive ion mode and multiple reaction monitoring was used as the detection system. Linearity was observed in the following ranges: 0.2-600 and 0.5-250 ng/mL for analysis of total and unbound concentrations of the tramadol enantiomers, respectively, and 0.1-300 and 0.25-125 ng/mL for total and unbound concentrations of the M1 and M2 enantiomers, respectively. The lower limits of quantitation were 0.2 and 0.5 ng/mL for analysis of total and unbound concentration of each tramadol enantiomer, respectively, and 0.1 and 0.25 ng/mL for total and unbound concentrations of M1 and M2 enantiomers, respectively. Intra- and interassay reproducibility and inaccuracy did not exceed 15%. Clinical application of the method to patients with neuropathic pain showed plasma accumulation of (+)-tramadol and (+)-M2 after a single oral dose of racemic tramadol. Fractions unbound of tramadol, M1 or M2 were not enantioselective in the patients investigated. (C) 2011 Elsevier B.V. All rights reserved.

Cooking Effects on Iron and Proteins Content of Beans (Phaseolus Vulgaris L.) by GF AAS and MALDI-TOF MS

The effects of domestic cooking on proteins, organic compounds and Fe distribution in beans (Phaseolus vulgaris L.) were investigated. Sequential extraction with different extractant solutions (mixture of methanol and chloroform 1:2 v/v, water, 0.5 mol L-1 NaCl, 70% v/v ethanol and 0.5 mol L-1 NaOH) were used for extracting lipids, albumins, globulins, prolamins and glutelins, respectively. Iron determination by graphite furnace atomic absorption spectrometry (GF AAS), proteins by Bradford method and organic compounds by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were carried out in this work. High concentration of albumins, globulins and glutelins were found in raw beans, while in the cooked beans, albumins and glutelins are main proteins types. The MALDI-TOF MS spectra of raw and cooked beans revealed that the domestic cooking altered the molecular weight of the organic compounds, since that in the cooked beans were found compounds between 2 and 3.5 kDa, which were not presented in the raw beans. Besides this, in cooked beans were also observed the presence of four compounds of high molecular weight (12-16 kDa), being that in the raw grains there is only one (ca. 15.2 kDa). In raw grains is possible to observe that Fe is mainly associated to albumins, globulins and glutelins. For cooked grains, Fe is associated to albumins and globulins.

Quantitative Proteomic Analysis of the Effect of Fluoride on the Acquired Enamel Pellicle

The acquired enamel pellicle (AEP) is a thin film formed by the selective adsorption of salivary proteins onto the enamel surface of teeth. The AEP forms a critical interface between the mineral phase of teeth (hydroxyapatite) and the oral microbial biofilm. This biofilm is the key feature responsible for the development of dental caries. Fluoride on enamel surface is well known to reduce caries by reducing the solubility of enamel to acid. Information on the effects of fluoride on AEP formation is limited. This study aimed to investigate the effects of fluoride treatment on hydroxyapatite on the subsequent formation of AEP. In addition, this study pioneered the use of label-free quantitative proteomics to better understand the composition of AEP proteins. Hydroxyapatite discs were randomly divided in 4 groups (n = 10 per group). Each disc was exposed to distilled water (control) or sodium fluoride solution (1, 2 or 5%) for 2 hours. Discs were then washed and immersed in human saliva for an additional 2 hours. AEP from each disc was collected and subjected to liquid chromatography electrospray ionization mass spectrometry for protein identification, characterization and quantification. A total of 45 proteins were present in all four groups, 12 proteins were exclusively present in the control group and another 19 proteins were only present in the discs treated with 5% sodium fluoride. Relative proteomic quantification was carried out for the 45 proteins observed in all four groups. Notably, the concentration of important salivary proteins, such as statherin and histatin 1, decrease with increasing levels of fluoride. It suggests that these proteins are repulsed when hydroxyapatite surface is coated with fluoride. Our data demonstrated that treatment of hydroxyapatite with fluoride (at high concentration) qualitatively and quantitatively modulates AEP formation, effects which in turn will likely impact the formation of oral biofilms.

Quantum Monte Carlo study of small aluminum clusters Al-n (n=2-13)

Using fixed node diffusion quantum Monte Carlo (FN-DMC) simulations and density functional theory (DFT) within the generalized gradient approximations, we calculate the total energies of the relaxed and unrelaxed neutral, cationic, and anionic aluminum clusters, Al-n (n = 1-13). From the obtained total energies, we extract the ionization potential and electron detachment energy and compare with previous theoretical and experimental results. Our results for the electronic properties from both the FN-DMC and DFT calculations are in reasonably good agreement with the available experimental data. A comparison between the FN-DMC and DFT results reveals that their differences are a few tenths of electron volt for both the ionization potential and the electron detachment energy. We also observe two distinct behaviors in the electron correlation contribution to the total energies from smaller to larger clusters, which could be assigned to the structural transition of the clusters from planar to three-dimensional occurring at n = 4 to 5.

Administration of a murine diet supplemented with conjugated linoleic acid increases the expression and activity of hepatic uncoupling proteins

Daily intake of conjugated linoleic acid (CLA) has been shown to reduce body fat accumulation and to increase body metabolism; this latter effect has been often associated with the up-regulation of uncoupling proteins (UCPs). Here we addressed the effects of a CLA-supplemented murine diet (similar to 2 % CLA mixture, cis-9, trans-10 and trans-10, cis-12 isomers; 45 % of each isomer on alternating days) on mitochondrial energetics, UCP2 expression/activity in the liver and other associated morphological and functional parameters, in C57BL/6 mice. Diet supplementation with CLA reduced both lipid accumulation in adipose tissues and triacylglycerol plasma levels, but did not augment hepatic lipid storage. Livers of mice fed a diet supplemented with CLA showed high UCP2 mRNA levels and the isolated hepatic mitochondria showed indications of UCP activity: in the presence of guanosine diphosphate, the higher stimulation of respiration promoted by linoleic acid in mitochondria from the CLA mice was almost completely reduced to the level of the stimulation from the control mice. Despite the increased generation of reactive oxygen species through oxi-reduction reactions involving NAD(+)/NADH in the Krebs cycle, no oxidative stress was observed in the liver. In addition, in the absence of free fatty acids, basal respiration rates and the phosphorylating efficiency of mitochondria were preserved. These results indicate a beneficial and secure dose of CLA for diet supplementation in mice, which induces UCP2 overexpression and UCP activity in mitochondria while preserving the lipid composition and redox state of the liver.

Nonculture-based identification of bacteria in milk by protein fingerprinting

Traditional methods for bacterial identification include Gram staining, culturing, and biochemical assays for phenotypic characterization of the causative organism. These methods can be time-consuming because they require in vitro cultivation of the microorganisms. Recently, however, it has become possible to obtain chemical profiles for lipids, peptides, and proteins that are present in an intact organism, particularly now that new developments have been made for the efficient ionization of biomolecules. MS has therefore become the state-of-the-art technology for microorganism identification in microbiological clinical diagnosis. Here, we introduce an innovative sample preparation method for nonculture-based identification of bacteria in milk. The technique detects characteristic profiles of intact proteins (mostly ribosomal) with the recently introduced MALDI SepsityperTM Kit followed by MALDI-MS. In combination with a dedicated bioinformatics software tool for databank matching, the method allows for almost real-time and reliable genus and species identification. We demonstrate the sensitivity of this protocol by experimentally contaminating pasteurized and homogenized whole milk samples with bacterial loads of 10(3)-10(8) colony-forming units (cfu) of laboratory strains of Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus. For milk samples contaminated with a lower bacterial load (104 cfu mL-1), bacterial identification could be performed after initial incubation at 37 degrees C for 4 h. The sensitivity of the method may be influenced by the bacterial species and count, and therefore, it must be optimized for the specific application. The proposed use of protein markers for nonculture-based bacterial identification allows for high-throughput detection of pathogens present in milk samples. This method could therefore be useful in the veterinary practice and in the dairy industry, such as for the diagnosis of subclinical mastitis and for the sanitary monitoring of raw and processed milk products.

The basic antioxidant structure for flavonoid derivatives

An antioxidant structure-activity study is carried out in this work with ten flavonoid compounds using quantum chemistry calculations with the functional of density theory method. According to the geometry obtained by using the B3LYP/6-31G(d) method, the HOMO, ionization potential, stabilization energies, and spin density distribution showed that the flavonol is the more antioxidant nucleus. The spin density contribution is determinant for the stability of the free radical. The number of resonance structures is related to the pi-type electron system. 3-hydroxyflavone is the basic antioxidant structure for the simplified flavonoids studied here. The electron abstraction is more favored in the molecules where ether group and 3-hydroxyl are present, nonetheless 2,3-double bond and carbonyl moiety are facultative.

Study of photorespiration in marine microalgae through the determination of glycolic acid using hydrophilic interaction liquid chromatography

Determination of organic acids in intracellular extracts and in the cultivation media of marine microalgae aid investigations about metabolic routes related to assimilation of atmospheric carbon by these organisms, which are known by their role in the carbon dioxide sink. The separation of these acids was investigated by hydrophilic interaction liquid chromatography (HILIC) using isocratic elution with a mobile phase composed of 70: 30 v/v acetonitrile/20 mmol/L ammonium acetate buffer (pH 6.8) and detection at 220 nm. HILIC allowed the determinations of glycolic acid, the most important metabolite for the evaluation of the photorespiration process in algae, to be made with better selectivity than that achieved by reversed phase liquid chromatography, but with less detectability. The concentration of glycolic acid was determined in the cultivation media and in intracellular extracts of the algae Tetraselmis gracilis and Phaeodactylum tricornutum submitted to different conditions of aeration: (i) without forced aeration, (ii) aeration with atmospheric air, and (iii) bubbling with N(2). The concentration of glycolic acid had a higher increase as the cultures were aerated with nitrogen, showing higher photorespiratory flux than that occurring in the cultures aerated with atmospheric air.

Amphetamine, cocaine and cannabinoids use among truck drivers on the roads in the State of Sao Paulo, Brazil

Drugs are important risk factors for traffic accidents. In Brazil, truck drivers report using amphetamines to maintain their extensive work schedule and stay awake. These drugs can be obtained without prescription easily on Brazilian roads. The use of these stimulants can result in health problems and can be associated with traffic accidents. There are Brazilian studies that show that drivers use drugs. However, these studies are questionnaire-based and do not always reflect real-life situations. The purpose of this study was to demonstrate the prevalence of drug use by truck drivers on the roads of Sao Paulo State, Brazil, during 2009. Drivers of large trucks were randomly stopped by police officers on the interstate roads during morning hours. After being informed of the goals of the study, the drivers gave written informed consent before providing a urine sample. In addition, a questionnaire concerning sociodemographic characteristics and health information was administered. Urine samples were screened for amphetamines, cocaine, and cannabinoids by immunoassay and the confirmation was performed using gas chromatography-mass spectrometry (GC-MS). Of the 488 drivers stopped, 456 (93.4%) provided urine samples, and 9.3% of them (n = 42) tested positive for drugs. Amphetamines were the most commonly found (n = 26) drug, representing 61.9% of the positive samples. Ten cases tested positive for cocaine (23.8%), and five for cannabinoids (11.9%). All drivers were male with a mean age of 40 +/- 10.8 years, and 29.3% of them reported some health problem (diabetes, high blood pressure and/or stress). A high incidence of truck drivers who tested positive for drug use was found, among other reported health problems. Thus, there is an evident need to promote a healthier lifestyle among professional drivers and a need for preventive measures aimed at controlling the use of drugs by truck drivers in Brazil. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

A Combined Study Using Ligand-Based Design, Synthesis, and Pharmacological Evaluation of Analogues of the Acetaminophen Ortho-Regioisomer with Potent Analgesic Activity

A ligand-based drug design study was performed to acetaminophen regioisomers as analgesic candidates employing quantum chemical calculations at the DFT/B3LYP level of theory and the 6-31G* basis set. To do so, many molecular descriptors were used such as highest occupied molecular orbital, ionization potential, HO bond dissociation energies, and spin densities, which might be related to quench reactivity of the tyrosyl radical to give N-acetyl-p-benzosemiquinone-imine through an initial electron withdrawing or hydrogen atom abstraction. Based on this in silico work, the most promising molecule, orthobenzamol, was synthesized and tested. The results expected from the theoretical prediction were confirmed in vivo using mouse models of nociception such as writhing, paw licking, and hot plate tests. All biological results suggested an antinociceptive activity mediated by opioid receptors. Furthermore, at 90 and 120 min, this new compound had an effect that was comparable to morphine, the standard drug for this test. Finally, the pharmacophore model is discussed according to the electronic properties derived from quantum chemistry calculations.

Oxidation of lysergic acid diethylamide (LSD) by peroxidases: a new metabolic pathway

Lysergic acid diethylamide (LSD) is a potent hallucinogen that is primarily metabolized to 2-oxo-3-hydroxy-LSD (O-H-LSD) and N-desmethyl-LSD (nor-LSD) by cytochrome P450 complex liver enzymes. Due to its extensive metabolism, there still is an interest in the identification of new metabolites and new routes of its metabolism in humans. In the present study, we investigated whether LSD could be a substrate for horseradish peroxidase or myeloperoxidase (MPO). Using liquid chromatography coupled to UV detection and electrospray ionization mass spectrometry (LC-UV-ESI-MS), we found that both peroxidases were capable of metabolizing LSD to the same compounds that have been observed in vivo (i.e., O-H-LSD and nor-LSD). In addition, we found another major metabolite, N,N-diethyl-7-formamido-4-methyl-6-oxo-2,3,4,4a,5,6-hexahydrobenzo[f]quinoline-2-carboxamide (FOMBK), which is an opened indolic ring compound. Hydrolysis of FOMBK led to the deformylated compound 7-amino-N,N-diethyl-4-methyl-6-oxo-2,3,4,4a,5,6-hexahydrobenzo[f]quinoline-2-carboxamide. The reactions of LSD with the peroxidases were chemiluminescent and sensitive to inhibition by reactive oxygen scavengers, which indicated that the classic peroxidase cycle is involved in this new alternative metabolic pathway. Considering that MPO is abundant in immune cells and also present in the central nervous system, the degradation pathway described in this study suggests a possible route of LSD metabolism that may occur concurrently with the in vivo reaction catalyzed by the cytochrome P450 system.

STUDY BY MASS SPECTROMETRY OF SOLUTIONS OF [HYDROXY(TOSYLOXY)IODO]BENZENE: PROPOSED DISPROPORTIONATION MECHANISMS

STUDY BY MASS SPECTROMETRY OF SOLUTIONS OF [HYDROXY(TOSYLOXY)IODO]BENZENE: PROPOSED DISPROPORTIONATION MECHANISMS. Solutions of [hydroxy(tosyloxy)iodo]benzene (HTIB or Koser's reagent) in acetonitrile were analyzed using high resolution electrospray ionization mass spectrometry (ESI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) under different conditions. Several species were characterized in these analyses. Based on these data, mechanisms were proposed for the disproportionation of the iodine(III) compounds in iodine(V) and iodine(I) species.