Strategy for choosing extraction procedures for NMR-based metabolomic analysis of mammalian cells

Metabolomic analysis of mammalian cells can be applied across multiple fields including medicine and toxicology. It requires the acquisition of reproducible, robust, reliable, and homogeneous biological data sets. Particular attention must be paid to the efficiency and reliability of the extraction procedure. Even though a number of recent studies have dealt with optimizing a particular protocol for specific matrices and analytical techniques, there is no universal method to allow the detection of the entire cellular metabolome. Here, we present a strategy for choosing extraction procedures from adherent mammalian cells for the global NMR analysis of the metabolome. After the quenching of cells, intracellular metabolites are extracted from the cells using one of the following solvent systems of varying polarities: perchloric acid, acetonitrile/water, methanol, methanol/water, and methanol/chloroform/water. The hydrophilic metabolite profiles are analysed using ¹H nuclear magnetic resonance (NMR) spectroscopy. We propose an original geometric representation of metabolites reflecting the efficiency of extraction methods. In the case of NMR-based analysis of mammalian cells, this methodology demonstrates that a higher portion of intracellular metabolites are extracted by using methanol or methanol/chloroform/water. The preferred method is evaluated in terms of biological variability for studying metabolic changes caused by the phenotype of four different human breast cancer cell lines, showing that the selected extraction procedure is a promising tool for metabolomic and metabonomic studies of mammalian cells. The strategy proposed in this paper to compare extraction procedures is applicable to NMR-based metabolomic studies of various systems.     

Development and validation of a confirmatory method for the determination of 12 non steroidal anti-inflammatory drugs in milk using liquid chromatography-tandem mass spectrometry

A rapid and reliable LC-MS/MS method for the simultaneous confirmation of twelve non steroidal anti-inflammatory drugs (NSAIDs) in bovine milk was developed and fully validated in accordance with the European Commission Decision 2002/657/EC. The validation scheme was built in accordance with the MRLs or target analytical levels (EU-CRL recommended concentrations and detection capabilities) of the analytes, except for diclofenac for which the lower level of validation achieved was 0.5 μg kg(-1) whereas its MRL is 0.1 μg kg(-1). The NSAIDs investigated were as follows: phenylbutazone (PBZ), oxyphenylbutazone (OPB), naproxen (NP), mefenamic acid (MF), vedaprofen (VDP), flunixin (FLU), 5-hydroxyflunixin (FLU-OH), tolfenamic acid (TLF), meloxicam (MLX), diclofenac (DC), carprofen (CPF) and ketoprofen (KTP). Several extraction procedures had been investigated during the development phase. Finally, the best results were obtained with a procedure using only methanol as the extraction solvent, with an evaporation step included and no further purification. Chromatographic separation was achieved on a C18 analytical column and the run was split in 2 segments. Matrix effects were also investigated. Data acquisition implemented for the confirmatory purpose was performed by monitoring 2 MRM transitions per analyte under the negative electrospray mode. Mean relative recoveries ranged from 94.7% to 110.0%, with their coefficients of variation lying between 2.9% and 14.7%. Analytical limits expressed in terms of decision limits (CCα) were evaluated between 0.69 μg kg(-1) (FLU) and 27.54 μg kg(-1) (VDP) for non-MRL compounds, and at 0.10 (DC), 15.37 (MLX), 45.08 (FLU-OH), and 62.96 μg kg(-1) (TLF) for MRL compounds. The validation results proved that the method is suitable for the screening and confirmatory steps as implemented for the French monitoring plan for NSAID residue control in bovine milk.     

Palladium-catalyzed coupling of N-tosylhydrazones with ortho substituted aryl halides: synthesis of 4-arylchromenes and related heterocycles

A convenient and efficient procedure for the synthesis of 4-arylchromenes, thiochromenes, and related heterocycles via a four step-sequence has been developed. The first three steps, which involve hydration of alkynes, hydrazones formation, and their Pd-coupling with ortho substituted aryl halides, furnished stereoselectively Z-trisubstituted olefins without any purification of the intermediates generated in each stage. These latter proved to be suitable precursors, in the last step, for the synthesis of the desired heterocycles of biological interest.     

Surface modification of poly(butylene terephthalate) nonwoven by photochemistry and biofunctionalization with peptides for blood filtration

The surface of meltblown poly(butylene terephthalate) (PBT) nonwoven was modified by photochemistry using the photolinker O‐succinimidyl 4‐azido‐2,3,5,6‐tetrafluorobenzoate for the introduction of activated ester functions and then coupling of molecular probes or biomolecules. Approximately 4000 pmol of (L )‐4,5‐[³H]‐lysine was fixed per PBT sample (1.13 cm²) and measured by liquid scintillation counting. The method consisted in a two‐step process: (a) coating of the clip (0.05 mg/sample) on the fibrous surface of the PBT followed by UV irradiation (30 min, 254 nm) and (b) coupling of amine‐terminated molecules (10^?3 M in phosphate buffer–CH₃CN [1/1, v/v], 20 h). Moreover, about 2000 pmol of ³H‐lysine can be immobilized on the PBT surface after UV irradiation (without clip) by aminolysis reactions with the created oxygenated functions. The derivatizations (via the clip and UV irradiation only) were stable after long‐term heating at 100 °C in water or under steam‐sterilization conditions. They induce neither modifications of the nonwoven morphology nor cytotoxicity. This method was applied for the grafting of peptides Gly‐Arg‐Gly‐Asp‐Ser and Gly‐Gly‐Gly‐Gly‐Gly to perform blood filtration experiments and to retain the leukocytes. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Tilcotil® Studies [3+2] additions with isothiazol-3(2H)-one 1,1-dioxide

Derivatives of isothiazol-3(2H)-one 1,1-dioxide react regiospecifically with 1,3-dipolar agents. The main regiocontrolling factor is ascertained to be the C?O group of the dipolarophile. The topology of the adducts is also in general agreement with predictions based on perturbation theory. Several adducts can be aromatized to heterocyclic equivalents of saccharin, and may then be elaborated into structural analogs of tenoxicam (Tilcotil® and piroxicam (Feldene®)).     

For a fixed Turaev shadow Jones-Vassiliev invariants depend polynomially on the gleams

We use Turaev's technique of shadows and gleams to parametrize the set of all knots in S ³ with the same Hopf projection. We show that the Vassiliev invariants arising from the Jones polynomial J _t (K) are polynomials in the gleams, i.e., for , the n-th order Vassiliev invariant u _n , defined by , is a polynomial of degree 2n in the gleams. Received: April 30, 1996     

Dediazoniations of Arene Diazonium Ions in Homogenous Solution. Part IV: Change-over from a heterolytic to a homolytic mechanism in 2,2,2-trifluoroethanol pyridine mixtures

There are only two dediazoniation products of benzenediazonium tetrafluoroborate in 2,2,2-trifluoroethanol (TFE), namely phenyl 2,2,2-trifluoroethyl ether ( 1 ) and fluorobenzene ( 2 ). The reaction kinetics are strictly first-order with respect to the diazonium salt. The addition of increasing amounts of pyridine to the system results in a gradual decrease in the yields of 1 and 2 and an increase in the yields of the homolytically formed products, benzene ( 3 ), biphenyl ( 4 ), isomeric phenylpyridines ( 5 ) and diazo tar ( 6 ). The reaction kinetics show that the rate of dediazoniation of the benzene diazonium salt increases with increasing amounts of pyridine. The reaction with added pyridine is no longer first-order with respect to the diazonium ion. The product analyses and the kinetic data are consistent with the view that in pure TFE this diazonium salt decomposes completely by a heterolytic mechanism. The addition of pyridine brings about a competitive homolytic mechanism which becomes increasingly dominant as the concentration of pyridine increases.     

Zinc diffusion in GaAs and zinc-induced disordering of GaAs/AlGaAs multiple quantum wells: a multitechnique study

A review of experimental results obtained by different techniques is presented on the problem of zinc diffusion. Zinc diffusion was carried out on Si-doped GaAs (n10¹⁸ cm^–3) and on multiple quantum well (MQW) structures. The samples were investigated by secondary-ion mass spectroscopy (SIMS), different imaging modes of scanning electron microscopy such as secondary electrons, cathodoluminescence (CL) and electron beam-induced current (EBIC), transmission electron microscopy on a wedge-shaped specimen (WTEM) and by photoluminescence (PL). A nonexponential decay of the low-temperature EBIC signal accompanied by a very low CL signal due to the high density of nonradiative recombination centres were observed in the diffused region of the n-doped GaAs. Indeed, PL measurements demonstrate that Ga vacancies play a key role on the mechanism of the Zn diffusion. On the impurity-induced disordered (IID) MQW samples, an enrichment of Al at the surface was observed by SIMS and confirmed by WTEM and PL. Low-temperature PL spectra show the gradual disappearance of the MQW excitonic transitions as the number of disordered layers increases. When all of the MQW structure is destroyed, the band-to-band recombinations in the IID produced alloy dominate the PL spectrum.     

Dediazoniations of Arene Diazonium Ions in Homogeneous Solution. Part III: Heterolytic arylations in 2,2,2-trifluoroethanol - an SN1-or an SN2-reaction?

Benzenediazonium tetrafluoroborate in 2,2,2-trifluoroethanol decomposes to give fluorobenzene and phenyl 2,2,2-trifluoroethyl ether. In the presence of benzene, toluene, trifluoromethyl-benzene or anisole, the respective biphenyl derivatives are formed in addition to fluorobenzene and the ether. The distribution of the isomeric substituted biphenyls is consistent with an electrophilic substitution. No homolytic products (diazo tars, benzene) are formed. The reaction kinetics clearly show that ether formation and aryl-dediazoniations are of second-order type, i.e. that trifluoroethanol and the benzene derivatives mentioned above are rate-determining factors. It is shown that these results exclude the S_N1-mechanism which is usually assumed for heterolytic dediazoniations; free aryl cations are therefore not involved in these reactions. An S_N2-like mechanism seems to be the most likely, but one involving an encounter complex containing the dissociated benzenediazonium ion is also consistent with the experimental data.     

Structural characterization by both positive and negative electrospray ion trap mass spectrometry of oligogalacturonates purified by high-performance anion-exchange chromatography

The off-line coupling of high-performance anion-exchange chromatography to electrospray ion trap mass spectrometry (ESI-IT-MS) is described. Two sets of isocratic conditions were optimised for the semi-preparative purification of oligogalacturonates of degree of polymerisation from 4 to 6 by monitoring eluates with either pulsed amperometric detection or evaporative light scattering detection in the presence of an online Dionex Carbohydrate Membrane Desalter (CMD). In these conditions, purified oligogalacturonate solutions were suitable, without further desalting steps, for infusion ESI-IT-MS experiments. This paper provides some interesting features of positive and negative ESI-IT-multiple MS (MSn) of these acidic oligosaccharides. The spectra acquired in both ion modes show characteristic fragments resulting from glycosidic bond and cross-ring cleavages. Under negative ionization conditions, the fragmentation of the singly-charged [M-H]- ions, as well as the Ci-, and Zi-, fragment ions through sequential MSn experiments, was always dominated by product ions from C- and Z-type glycosidic cleavages. All spectra also displayed 0.2 A-type cross-ring cleavage ions which carry linkage information. Collision-induced dissociation (CID) spectra of sodium-cationized species obtained under positive ionization conditions were more complex. Successive MSn experiments also led to the 0.2 A-type cross-ring cleavage ions observed together with B- and Y-type ions. The presence of the 0.2 A ion series was related to Mr 60 (C2H4O2) losses. Combined with the absence of the Mr 30 (CH2O) and the Mr 90 (C3H6O3) ions, these ions were indicative of 1-4 type glycosidic linkage.