Fluorescence probes to detect lipid-derived radicals.

Lipids and their metabolites are easily oxidized in chain reactions initiated by lipid radicals, forming lipid peroxidation products that include the electrophiles 4-hydroxynonenal and malondialdehyde. These markers can bind cellular macromolecules, causing inflammation, apoptosis and other damage. Methods to detect and neutralize the initiating radicals would provide insights into disease mechanisms and new therapeutic approaches. We describe the first high-sensitivity, specific fluorescence probe for lipid radicals, 2,2,6-trimethyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)-6-pentylpiperidine-1-oxyl (NBD-Pen). NBD-Pen directly detected lipid radicals in living cells by turn-on fluorescence. In a rat model of hepatic carcinoma induced by diethylnitrosamine (DEN), NBD-Pen detected lipid radical generation within 1 h of DEN administration. The lipid radical scavenging moiety of NBD-Pen decreased inflammation, apoptosis and oxidative stress markers at 24 h after DEN, and liver tumor development at 12 weeks. Thus, we have developed a novel fluorescence probe that provides imaging information about lipid radical generation and potential therapeutic benefits in vivo.

Simultaneous determination of nicotine, cotinine, and nicotine N-oxide in human plasma, semen, and sperm by LC-Orbitrap MS.

Nicotine (Nic) distribution in human fluids and tissues has a deleterious effect on human health. In addition to its poisoning profile, Nic may contribute to the particular impact of smoking on human reproduction. Although present in seminal fluid, still nobody knows whether nicotine is available in sperm or not. Herein, we developed and validated a new bioanalytical method, for simultaneous determination of Nic, cotinine (Cot), and nicotine N'-oxide (Nox) in human plasma, semen, and sperm by LC-ESI-orbitrap-MS. Blood and semen samples were collected from 12 healthy smoking volunteers in this study. Sperm bodies were then separated quantitatively from 1 mL of semen samples by centrifugation. The developed method was fully validated for plasma following European and American guidelines for bioanalytical method validation, and partial validation was applied to semen analysis. Plasma, semen, and sperm samples were treated by trichloroacetic acid solution for protein direct precipitation in single extraction step. The established calibration range for Nic and Nox in plasma and semen was linear between 5 and 250 ng/mL, and for Cot between 10 and 500 ng/mL. Nic and Cot were detected in human sperm at concentrations as high as in plasma. In addition, Nox was present in semen and sperm but not in plasma. Graphical abstract Nicotine correlation between plasma and semen a; Nicotine correlation between semen and sperm c; Cotinine correlation between plasma and semen b; Cotinine correlation between semen and sperm d.

Intracerebral antioxidant ability of mature rats after neonatal hypoxic-ischemic brain injury estimated using the microdialysis-electron spin resonance method.

AIM: The intracerebral antioxidant ability of mature rats after neonatal hypoxic-ischemic (HI) brain injury was estimated using the microdialysis-electron spin resonance method. MATERIAL AND METHODS: Seven-day-old Wistar rats were subjected to a modified Levine's procedure for producing HI brain injury. After HI insult, pups were returned and reared with their dams. Seven weeks after HI insult, their intracerebral antioxidant abilities were measured using the microdialysis-electron spin resonance method after the intraperitoneal injection of 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl. Ascorbic acid, L-cysteine, and glutathione (GSH) were also determined. The rats without HI insult were used as a control. RESULTS: The decay rate of 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl in the non-ligated side of the cerebral hemisphere of the HI group was significantly larger than that of the control group. The amounts of ascorbic acid in the perfusate from the non-ligated side of the HI group were about four times larger than those of the control group. The amounts of L-cysteine and GSH of the HI group were about 10 times larger than those of the control group. CONCLUSIONS: The antioxidant ability in the non-ligated sides of the cerebral hemispheres of the mature rats 7 weeks after neonatal HI insult was higher than that of the control group. Higher amounts of ascorbic acid and GSH supported the higher antioxidant ability. The increase of the intracerebral antioxidant ability of the non-ligated side indicates the compensation of motor function for the lost side. The present results should offer important insights into the prognosis for hypoxic-ischemic encephalopathy.

Stability indicating RP-HPLC method development and validation for the simultaneous determination of aminexil and minoxidil in pharmaceutical dosage form.

The objective of the present work is to develop stability indicating high-performance liquid chromatographic method for the simultaneous determination of aminexil and minoxidil in pharmaceutical dosage form. The chromatographic separation was achieved with BDS Hypersil C18 column (250 mm×4.6 mm×5 µ) as stationary phase and phosphate buffer and acetonitrile (78:22) as mobile phase. The method was employed by using a flow rate of 1.1 mL/min kept at 30°C. The detection wavelength was kept at 238 nm by using photo-diode array detector. The retention times of the aminexil and minoxidil were found to be 2.3 min and 3.9 min, respectively. The method developed was validated in accordance with ICH guidelines with respect to the stability indicating capacity of the method including system suitability, accuracy, precision, linearity, range, limit of detection, limit of quantification and robustness. The linearity responses of aminexil and minoxidil were found to be in the concentration ranges of 18.75-112.5 µg/mL and 25-150 µg/mL, respectively. The LOD and LOQ values for aminexil were found to be 0.31 and 0.92 µg/mL and minoxidil were found to be 0.03 and 0.10 µg/mL respectively. The percentage recoveries for both the drugs were found in the range of 98-101%. This method is accurate, precise and sensitive; hence, it can be employed for routine quality control of aminexil and minoxidil in pharmaceutical industries and drug testing laboratories.

Communication: Orientational self-ordering of spin-labeled cholesterol analogs in lipid bilayers in diluted conditions.

Lipid-cholesterol interactions are responsible for different properties of biological membranes including those determining formation in the membrane of spatial inhomogeneities (lipid rafts). To get new information on these interactions, electron spin echo (ESE) spectroscopy, which is a pulsed version of electron paramagnetic resonance (EPR), was applied to study 3ß-doxyl-5α-cholestane (DCh), a spin-labeled analog of cholesterol, in phospholipid bilayer consisted of equimolecular mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine. DCh concentration in the bilayer was between 0.1 mol.% and 4 mol.%. For comparison, a reference system containing a spin-labeled 5-doxyl-stearic acid (5-DSA) instead of DCh was studied as well. The effects of "instantaneous diffusion" in ESE decay and in echo-detected (ED) EPR spectra were explored for both systems. The reference system showed good agreement with the theoretical prediction for the model of spin labels of randomly distributed orientations, but the DCh system demonstrated remarkably smaller effects. The results were explained by assuming that neighboring DCh molecules are oriented in a correlative way. However, this correlation does not imply the formation of clusters of cholesterol molecules, because conventional continuous wave EPR spectra did not show the typical broadening due to aggregation of spin labels and the observed ESE decay was not faster than in the reference system. So the obtained data evidence that cholesterol molecules at low concentrations in biological membranes can interact via large distances of several nanometers which results in their orientational self-ordering.

Ability of water-soluble biosubstances to eliminate hydroxyl and superoxide radicals examined by spin-trapping ESR measurements: two-dimensional presentation of antioxidative ability.

The hydroxyl- and superoxide-radical-eliminating ability of water-soluble biosubstances was examined by ESR combined with the spin-trapping method, indicating a median inhibitory dose, ID(h)(50) (mM) and id(h)(50) (mg/mL) for the hydroxyl radical, and ID(s)(50) (mM) and id(s)(50) (mg/mL) for the superoxide radical. Both the 1/[ID(h)(50) (mM)] and 1/[ID(s)(50) (mM)] values of selected biosubstances were linearly related to the second-order rate constant, k(2) (M(-1) s(-1)), defined for the reaction between biosubstances and the radicals in a logarithmic presentation. The result indicates that ID(h)(50) (mM) and ID(s)(50) (mM) are suitable parameters for both types of radical-eliminating ability. The obtained results are depicted two-dimensionally, taking id(h)(50) (mg/mL) as the abscissa and id(s)(50) (mg/mL) as the ordinate in the ROS inhibitory diagram. The biosubstances tested were assigned to five separate areas characterized by their functional groups on the diagram. The obtained ROS inhibitory diagram indicates the possibility for screening appropriate antioxidants.

Sorption selectivity in natural organic matter studied with nitroxyl paramagnetic relaxation probes.

Sorption site selectivity and mechanism in natural organic matter (NOM) were addressed spectroscopically by the sorption of paramagnetic nitroxyl compounds (spin probes) of different polarity, TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) and HTEMPO (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl). The sorbents were Pahokee peat, Beulah-Zap lignite, and a polystyrene-poly(vinyl methyl ether) (PS-PVME) polymer blend representing the mixed aliphatic-aromatic, polar-nonpolar character of NOM. Nuclear-electron spin interaction serves as an efficient relaxation pathway, resulting in attenuation of the (13)C-CP/TOSS NMR signal for (13)C nuclei in proximity to the N-O· group (r(-6) dependence). In the natural solids the spin probes sorbed more specifically (greater isotherm nonlinearity) and had lower rotational mobility (broader electron paramagnetic resonance signals) than in PS-PVME. Titration with spin probe indicated almost no selectivity for the different carbon functional groups of PS-PVME, and little to no selectivity for the different carbon moieties of Pahokee and Beulah, including aromatic, alkyl, O-alkyl, di-O-alkyl, and O-methyl. In any case, sorption site selectivity of spin probes to NOM was always weaker than partition selectivity found in model solvent-water (toluene, hexadecane, anisole, octanol) and cellulose-water systems. The results indicate little or no preferential sorption in NOM based on functional group chemistry or putative microdomain character, but rather are consistent with the filling of pores whose walls have an average chemical environment reflecting the bulk chemical composition of the solid. This work demonstrates for the first time the use of paramagnetic probes to study sorption specificity.

Synthesis and preliminary conformational analysis of TOAC spin-labeled analogues of the medium-length peptaibiotic tylopeptin B.

A set of analogues of the 14-residue peptaibol tylopeptin B, containing the stable free-radical 4-amino-1-oxyl-2,2,6,6,-tetramethylpiperidine-4-carboxylic acid (TOAC) at one or two selected positions, was synthesized by the solid-phase methodology. A solution conformational analysis performed by FTIR absorption and CD suggests that, in membrane-mimicking solvents, the labeled tylopeptin B analogues preserve the helical propensity of the parent peptide, with a preference for the α-helix or the 3(10) -helix type depending upon the nature of the solvent. In aqueous environment, the spin-labeled analogues present a higher content of helical conformation as a consequence of the strong helix promoter effect of the conformationally constrained TOAC residue. We observed a progressive increase of the quenching effect of the nitroxyl radical on the fluorescence of the N-terminal tryptophan as TOAC replaces the Aib residue at positions 13, 8, and 4, respectively. A membrane permeabilization assay performed on two selected analogues, TOAC(8) - and TOAC(13) -tylopeptin B, showed that the labeled peptides exhibit membrane-modifying properties comparable with those of the natural peptaibiotic. We conclude that our TOAC paramagnetic analogues of tylopeptin B are good models for a detailed ESR investigation of the mechanism of membrane permeabilization induced by medium-length peptaibiotics.

Studies on log Po/w of quinoxaline di-N-oxides: a comparison of RP-HPLC experimental and predictive approaches.

As reported in our previous papers, a series of quinoxaline-2-carboxamide 1,4-di-N-oxide derivatives were synthesized and studied as anti-tuberculosis agents. Here, the capability of the shake-flask method was studied and the retention time (expressed as log K) of 20 compounds were determined by RP-HPLC analysis. We found that the prediction of log P by the RP-HPLC analysis can result in a high accuracy and can replace the shake-flask method avoiding the experimental problems presented by quinoxaline di-N-oxides. The studied compounds were subjected to the ALOGPS module with the aim of comparing experimental log P(o/w) values and predicted data. Moreover, a preliminary in silico screening of the QSAR relationship was made confirming the influence of reduction peak potential, lipophilicity, H-bond donor capacity and molecular dimension descriptors on anti-tuberculosis activity.

EPR imaging of sinapyl alcohol and its application to the study of plant cell wall lignification.

In bioimaging, bioorthogonal chemistry is most often used to visualize chemical reporters by fluorescence in their native environment. Herein, we show that TEMPO-based probes can be ligated to monolignol reporters by Diels-Alder chemistry in plant cell walls, paving the way for the study of lignification by EPR spectroscopy and imaging.

LC/MS analysis of three-dimensional model cells exposed to cigarette smoke or aerosol from a novel tobacco vapor product.

A novel tobacco vapor product (NTV) contains tobacco leaves and generates nicotine-containing aerosols using heating elements. Subchronic biological effects have been evaluated previously using three-dimensional bronchial epithelial model cells by repeated exposure to cigarette smoke (CS) and the NTV aerosols; however, the intracellular exposure characteristics have not been studied in detail. In this study, cells were initially exposed to an aqueous extract (AqE) of cigarette smoke (CS) at two concentration levels, and the cell lysate underwent untargeted analysis by LC-high resolution mass spectrometry to determine the exogenous compounds present in the cells. Among the thousands of peaks detected, four peaks showed a CS-dependency, which were reproducibly detected. Two of the peaks were nicotine and nicotine N-oxide, and the other two putative compounds were myosmine and norharman. The cells were then exposed to an AqE of CS in various combinations of exposure and post-exposure culture durations. Post-exposure culturing of cells with fresh medium markedly decreased the peak areas of the four compounds. The in-vitro switching study of CS to NTV aerosols was investigated by intermittently exposing cells to an AqE of CS four times, followed by exposure to either an AqE of CS, NTV aerosol or medium another four times. Switching to NTV reduced myosmine and norharman levels, which are known CS constituents. The results indicate that extracellular compounds inside cells reflect the exposure state outside cells. Thus, monitoring functional changes to cells in these exposure experiments is feasible.

Structural characterization of in vitro rat liver microsomal metabolites of antihistamine desloratadine using LTQ-Orbitrap hybrid mass spectrometer in combination with online hydrogen/deuterium exchange HR-LC/MS.

In vitro drug metabolism study is an integral part of drug discovery process. In this report, we have described the application of LTQ-Orbitrap hybrid mass spectrometer in conjunction with online hydrogen (H)/deuterium (D) exchange high resolution (HR)-LC/MS for structural characterization of in vitro rat liver microsomal metabolites of antihistamine desloratadine. Five metabolites M1--M5 have been identified, including three hydroxylated metabolites M1--M3, one N-oxide M4 and one uncommon aromatized N-oxide M5. Accurate mass data have been obtained in both full scan and MSn mode support assignments of metabolite structures with reported mass errors less than 3 ppm. Online H/D exchange HR-LC/MS experiments provide additional evidence in differentiating hydroxylated metabolites from N-oxides. This study demonstrates the effectiveness of this approach in structural characterization of drug metabolites.

Mechanistic insight into TEMPO-inhibited polymerisation: simultaneous determination of oxygen and inhibitor concentrations by EPR.

Convolution-based fitting of EPR spectra makes it possible to simultaneously determine concentrations of TEMPO and oxygen in TEMPO-inhibited polymerisations and autoxidations; this method is useful for understanding the chemistry of inhibitor mixtures.

Oxygen permeability and oxidative stability of fish oil-loaded electrosprayed capsules measured by Electron Spin Resonance: Effect of dextran and glucose syrup as main encapsulating materials.

The oxygen permeability and oxidative stability of fish oil-loaded electrosprayed capsules were studied by Electron Spin Resonance (ESR). Electrosprayed capsules with dextran as main biopolymer showed a significantly faster broadening (ΔHpp) of 16-doxyl-stearate ESR spectrum when compared to glucose syrup capsules. This finding indicates a higher oxygen permeability of dextran capsules than glucose syrup capsules, which is explained by a reduced average free volume in the glucose syrup matrix than in the dextran shell. Moreover, glucose syrup capsules showed a significantly lower increase in the peak-to-peak amplitude of N-tert-butyl-α-phenylnitrone (PBN) ESR spectrum during storage when compared to dextran capsules. This implies a higher oxidative stability of glucose syrup capsules than dextran capsules, which correlated well with the lower oxygen permeability of the former. These results indicated the importance of the oxygen barrier properties of the wall materials when encapsulating long chain omega-3 polyunsaturated fatty acids by electrospraying.

Safety assessment of food and herbal products containing hepatotoxic pyrrolizidine alkaloids: interlaboratory consistency and the importance of N-oxide determination.

INTRODUCTION: Two recent mass spectrometry-based reports concerning Senecio scandens yielded remarkably dissimilar pyrrolizidine alkaloid constituents. In both studies, and in a related analysis of Senecio scandens and Tussilago farfara using micellar electrokinetic chromatography, the presence of hazardous N-oxides of the alkaloids was either not considered or was inadequately considered. This raises concerns about the effectiveness of the methodologies used in these, and similar, studies in assessing the pyrrolizidine alkaloid content and the safety of food, food supplements and medicines for human use. OBJECTIVE: To highlight essential analytical requirements for confident assessment of pyrrolizidine alkaloid-related safety of food and herbal products for human use. METHODOLOGY: Direct infusion-ESI MS and HPLC-ESI MS were used to analyse samples derived from liquid-liquid partitioning experiments and from strong cation exchange, solid-phase extraction of pyrrolizidine alkaloids and their N-oxides. RESULTS: A simple solvent partitioning experiment using pure senecionine and senecionine-N-oxide, two constituents reported in one of the mass spectrometry-based studies of S. scandens, clearly demonstrated the inadequacy of the reported method to detect and quantitate hazardous pyrrolizidine alkaloid N-oxide components. A preliminary LCMS analysis of commercially-prepared extracts of comfrey roots (Symphytum officinale and S. uplandicum s. l.) was used as a model to highlight the analytical importance of N-oxides in the safety assessment of pyrrolizidine alkaloid-containing medicinal herbs. CONCLUSIONS: This study highlighted significant differences in the reported identification of pyrrolizidine alkaloids from the same plant species, and clearly demonstrated the inadequacy of some procedures to include N-oxides in the assessment of pyrrolizidine alkaloid-related safety of food and herbal products.

Paramagnetic Tag for Glycosylation Sites in Glycoproteins: Structural Constraints on Heparan Sulfate Binding to Robo1.

An enzyme- and click chemistry-mediated methodology for the site-specific nitroxide spin labeling of glycoproteins has been developed and applied. The procedure relies on the presence of single N-glycosylation sites that are present natively in proteins or that can be engineered into glycoproteins by mutational elimination of all but one glycosylation site. Recombinantly expressing glycoproteins in HEK293S (GnT1-) cells results in N-glycans with high-mannose structures that can be processed to leave a single GlcNAc residue. This can in turn be modified by enzymatic addition of a GalNAz residue that is subject to reaction with an alkyne-carrying TEMPO moiety using copper(I)-catalyzed click chemistry. To illustrate the procedure, we have made an application to a two-domain construct of Robo1, a protein that carries a single N-glycosylation site in its N-terminal domains. The construct has also been labeled with 15N at amide nitrogens of lysine residues to provide a set of sites that are used to derive an effective location of the paramagnetic nitroxide moiety of the TEMPO group. This, in turn, allowed measurements of paramagnetic perturbations to the spectra of a new high affinity heparan sulfate ligand. Calculation of distance constraints from these data facilitated determination of an atomic level model for the docked complex.

Electron paramagnetic spectroscopic evidence of exercise-induced free radical accumulation in human skeletal muscle.

The present study determined if acute exercise increased free radical formation in human skeletal muscle. Vastus lateralis biopsies were obtained in a randomized balanced order from six males at rest and following single-leg knee extensor exercise performed for 2 min at 50% of maximal work rate (WR(MAX)) and 3 min at 100% WR(MAX). EPR spectroscopy revealed an exercise-induced increase in mitochondrial ubisemiquinone (UQ*-) [0.167 +/- 0.055 vs. rest: 0.106 +/- 0.047 arbitrary units (AU)/g total protein (TP), P < 0.05] and alpha-phenyl-tert-butylnitrone-adducts (112 +/- 41 vs. rest: 29 +/- 9 AU/mg tissue mass, P < 0.05). Intramuscular lipid hydroperoxides also increased (0.320 +/- 0.263 vs. rest: 0.148 +/- 0.071 nmol/mg TP, P < 0.05) despite an uptake of alpha-tocopherol, alpha-carotene and beta-carotene. There were no relationships between mitochondrial volume density and any biomarkers of oxidative stress. These findings provide the first direct evidence for intramuscular free radical accumulation and lipid peroxidation following acute exercise in humans.

Investigation of the generation of hydroxyl radicals and their oxidative role in the presence of heterogeneous copper catalysts.

The presence and impact of hydroxyl radicals generated via the catalytic decomposition of H(2)O(2) over heterogeneous copper catalysts were investigated by using two detection methods, an electron spin resonance-spin trapping method and a chemical probe method. Detection of the (5,5-dimethyl-1-pyrroline-N-oxide)-OH adduct signal and formation of 4-chlorocatechol during the oxidation of a 4-chlorophenol substrate demonstrated that the three heterogeneous copper catalysts employed here (CuO, Cu/Al(2)O(3) and CuO.ZnO/Al(2)O(3)) were capable of generating hydroxyl radicals in combination with H(2)O(2). The oxidative mechanism of the hydroxyl radical in the presence of heterogeneous copper catalysts is discussed with regard to the further oxidation of the (5,5-dimethyl-1-pyrroline-N-oxide)-OH adduct and hydroxylated products of 4-chlorophenol oxidation. Interestingly, integration of the 5,5-dimethyl-1-pyrroline-N-oxide-OH adduct signal could not be used to reliably measure the total amount of hydroxyl radicals generated as a result of oxidative attack on the adduct. This may be as a result of locally higher hydroxyl radical concentrations in the presence of a heterogeneous catalyst leading to further unwanted oxidation of the (5,5-dimethyl-1-pyrroline-N-oxide)-OH.

Multipurpose EPR loop-gap resonator and cylindrical TE011 cavity for aqueous samples at 94 GHz.

A loop-gap resonator (LGR) and a cylindrical TE(011) cavity resonator for use at W band, 94 GHz, have been designed and characterized using the Ansoft (Pittsburgh, PA) high frequency structure simulator (HFSS; Version 10.0). Field modulation penetration was analyzed using Ansoft MAXWELL 3D (Version 11.0). Optimizing both resonators to the same sample sizes shows that EPR signal intensities of the LGR and TE(011) are similar. The 3 dB bandwidth of the LGR, on the order of 1 GHz, is a new advantage for high frequency experiments. Ultraprecision electric discharge machining (EDM) was used to fabricate the resonators from silver. The TE(011) cavity has slots that are cut into the body to allow penetration of 100 kHz field modulation. The resonator body is embedded in graphite, also cut by EDM techniques, for a combination of reasons that include (i) reduced microwave leakage and improved TE(011) mode purity, (ii) field modulation penetration, (iii) structural support for the cavity body, and (iv) machinability by EDM. Both resonators use a slotted iris. Variable coupling is provided by a three-stub tuning element. A collet system designed to hold sample tubes has been implemented, increasing repeatability of sample placement and reducing sample vibration noise. Initial results include multiquantum experiments up to 9Q using the LGR to examine 1 mM 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) in aqueous solution at room temperature and field modulation experiments using the TE(011) cavity to obtain an EPR spectrum of 1 microM TEMPO.