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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.

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.

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 (ΔH pp ) 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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 15 N 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.

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.

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.

2-Hydroxypyridine-1-oxide (HOPO) is utilized in the pharmaceutical sector to facilitate the formation of amide bonds. This present work describes the development of a HPLC high pH ion-pairing method for the trace analysis of HOPO in pharmaceutically relevant materials. Several method development aspects are discussed and the application of this method to several late-stage programs is presented., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (© 2014 The Authors. Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology.)

A derivatization LC-MS/MS method was developed and qualified for the trace level quantification of 2-hydroxypyridine N-oxide (HOPO). HOPO is a coupling reagent used in the syntheses of active pharmaceutical ingredients (APIs) to form amide bonds. HOPO was recently confirmed to generate a positive response in a GLP Ames bacterial-reverse-mutation test, classifying it as a mutagenic impurity and as such requiring its control in APIs to the threshold of toxicological concern (TTC). The derivatization reagent 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride) was used in a basic solution to convert HOPO into the corresponding dansyl-derivative. The derivative was separated from different APIs and reagents by liquid chromatography. The detection of the HOPO dansyl-derivative was achieved by mass spectrometry in selected reaction monitoring (SRM) mode. The LC-MS/MS method had a reporting limit of 0.1ng/mL HOPO, which corresponds to 0.1ppm HOPO relative to an API at 1mg/mL, and a linearity range of 0.1-25ng/mL HOPO analyte. Recoveries of HOPO standards spiked into three different API matrices at 0.2, 1.2, and 20ppm levels were all within 90-100%. An SRM-based confirmatory methodology using the ratios of two fragment ions at three CID energies was developed to verify the identity of HOPO when present at ≥0.6ppm. This identity confirmation can be employed to prevent potential false positive detection of mutagenic impurities at trace level. It can be broadly applicable for the confirmation of analytes when the analytes generate at least two major fragments in tandem mass spectrometry experiments., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

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 3β-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.

Chloroquine (CQ) (1) which has endured as one of the most powerful antimalarial drugs was subjected to oxidative stress conditions and the degradation profile was studied. The oxidative stress condition of CQ furnished one major degradation product along with other minor degradation products. The unknown major degradation product was identified in HPLC and pure impurity was isolated using column chromatography. The structure of this major product was elucidated using UV, FT-IR, (1)H NMR, (13)C NMR, 2D NMR (HSQC) and mass spectral data. Based on the results obtained from the different spectroscopic studies, it was confirmed that the N-oxide was formed at the tertiary amine nitrogen instead of the pyridine nitrogen. Subsequently, an efficient and simple synthetic approach was developed for the synthesis of chloroquine-N-oxide using a work-up procedure that does not require chromatography techniques for further purification. It was observed that the spectral data of the isolated degradation product coincided appropriately with the synthesized product spectral data., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

Antioxidative effects of sesamin (a mixture of sesamin and episesamin) were evaluated in the liver, kidney and inferior vena cava of living rats using a radiofrequency ESR method. TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl, was used as an in vivo redox probe, the half-life of which is believed to be correlated with the antioxidant status. The oral administration of sesamin (250 mg/kg rat weight) 3 h before ESR measurements shortened the half-life of TEMPOL in the liver by 10 - 15% as compared with the controls, but did not affect the other organs. This effect was maintained for at least 3 h after the administration, and then disappeared at 24 h, corresponding to the results of our preliminary pharmacokinetic studies. Changes in the reducing ability were observed only in the hepatic sites of the sesamin-treated rats. These findings suggest that sesamin exhibits effective antioxidant activity in the liver via modulation of the intracellular redox status related to TEMPOL reduction.

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.

The effects of different drying methods (40 °C conventional and vacuum oven, 25 °C ambient air and freeze dry) on the stability of two red wine grape (Pinot Noir, PN and Merlot, M) byproducts, pomace containing skins and seeds (P) and pomace containing skins only (S) were investigated. Freeze dried samples retained the highest bioactive compounds with total phenolic content (TPC) of 21.19-67.74 mg GAE/g d.m., anthocyanin content (ACY) of 0.35-0.76 mg Mal-3-glu/g d.m., DPPH antiradical scavenge activity (ARS) of 22.01-37.46 mg AAE/g d.m., and total flavanol content (TFC) of 30.16-106.61 mg CE/g d.m., followed with ambient air dried samples. All samples lost significant amount of bioactive compounds during 16 wk of storage at 15 ± 2 °C, in which ambient air and freeze dried samples had TPC reduction of 32-56% and 35-58%, respectively, but ARS in PN-P and M-P still remained more than 50 mg TE/g d.m. Overall, TPC, ARS, and TFC were higher in PN than in M, and higher in pomace than in skins, while reverse results were observed in ACY. Pomace extracts showed higher antibacterial efficiency against Listeria innocua ATCC 51142 than Escherichia coli ATCC 25922 with minimal inhibition concentration (MIC) of 3%, 6%, 4%, and 9% against E. coli, and 2%, 7%, 3%, and 8% against L. innocua for PN-P, PN-S, M-P, and M-S samples, respectively. Dietary fiber content of samples was 57-63% of total dry matter. This study demonstrated that Pinot Noir and Merlot pomace are good sources of antioxidant dietary fibers and may be incorporated into various food products as a functional ingredient. Practical Application:  Wine grape pomace (WGP), the byproduct of wine making, is a good source of polyphenols and dietary fibers and may be incorporated into various food products as a functional ingredient. This study reported the effect of four drying methods and storage at 15 ± 2 °C up to 4 months on the retention of polyphenols and antioxidant activity in two types of red WGP (with and without seeds). Antibacterial activity, dietary fiber content and the basic physicochemical properties of dried pomace powder were also reported. The information is essential for developing specific applications of the pomace., (© 2012 Institute of Food Technologists®)

A liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-ITMS) method was developed for the simultaneous analysis of strychnine, brucine and their major metabolites. Strychnine and brucine were individually incubated with rat liver S9 fraction. The incubation samples were pooled together and analyzed with LC-ESI-ITMS in positive ion and full-scan detection mode. The calibration curves of strychnine and brucine in rat liver showed good linearity in ranges of 0.020 to 8.0 µg/mL for strychnine and 0.020 to 8.5 µg/mL for brucine. The limits of detections were both 0.008 µg/mL and the recoveries were 88.3 and 83.2% for strychnine and brucine, respectively. Two metabolites were identified as strychnine N-oxide and brucine N-oxide by comparing the molecular mass, retention time, full-scan mass spectra, tandem MS and MS(3) spectra with those of strychnine and brucine. The developed method provided high sensitivity and selectivity for the determination of poisonous alkaloids and their major metabolites and can be applied in the determination of samples in forensic and clinically toxicological cases.

The synthesis of filibuvir, a hepatitis C virus polymerase inhibitor candidate, involves use of 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO), a potentially genotoxic free radical reagent. A headspace gas chromatographic method utilizing selected-ion monitoring (SIM) mode mass spectrometric detection was developed, validated and applied to the determination of low levels of TEMPO in filibuvir. The GC-MS method was validated in terms of specificity, linearity, precision, accuracy/recovery, limit of quantitation (LOQ) and limit of detection (LOD). The method was shown to be specific for detection of TEMPO in the presence of filibuvir and exhibited acceptable linearity (r ≥ 0.997) over the range of 4-60 ppm vs. filibuvir (0.4-6.0 μg/mL). The system precision was 14% and 8% relative standard deviation (RSD) at the 4 ppm and 8 ppm levels, respectively. Method repeatability was 15% and 13% RSD at the 4 ppm and 8 ppm levels, respectively. Recovery was approximately 50-80% across the method range. Accuracy was 135% and 91% vs. nominal at the 4 and 8 ppm levels, respectively. The LOQ and LOD are 4 ppm and 2 ppm, respectively. Thirteen batches of filibuvir drug substance had no detectable TEMPO (≤ 2 ppm). Purge studies demonstrated that the synthetic process has an extremely high capability to remove TEMPO and consistently delivers filibuvir drug substance with TEMPO levels well below the staged threshold of toxicological concern., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Pyrrolizidine alkaloid (PA)-containing plants are widely distributed in the world. PAs are hepatotoxic, affecting livestock and humans. PA N-oxides are often present together with PAs in plants and also exhibit hepatotoxicity but with less potency. HPLC-MS is generally used to analyze PA-containing herbs, although PA references are unavailable in most cases. However, to date, without reference standards, HPLC-MS methodology cannot distinguish PA N-oxides from PAs because they both produce the same characteristic ions in mass spectra. In the present study, the mass spectra of 10 PA N-oxides and the corresponding PAs were systemically investigated using HPLC-MS to define the characteristic mass fragment ions specific to PAs and PA N-oxides. Mass spectra of toxic retronecine-type PA N-oxides exhibited two characteristic ion clusters at m/z 118-120 and 136-138. These ion clusters were produced by three unique fragmentation pathways of PA N-oxides and were not found in their corresponding PAs. Similarly, the nontoxic platynecine-type PA N-oxides also fragmented via three similar pathways to form two characteristic ion clusters at m/z 120-122 and 138-140. Further application of using these characteristic ion clusters allowed successful and rapid identification of PAs and PA N-oxides in two PA-containing herbal plants. Our results demonstrated, for the first time, that these characteristic ion clusters are unique determinants to discriminate PA N-oxides from PAs even without the availability of reference samples. Our findings provide a novel and specific method to differentiate PA N-oxides from PAs in PA-containing natural products, which is crucial for the assessment of their intoxication., (Copyright © 2012 John Wiley & Sons, Ltd.)

Overhauser-enhanced MRI (OMRI) offers the potentiality of detecting low-concentrated species generated by specific biological processes. However molecular imaging applications of OMRI need significant improvement in spatial localization. Here it is shown that 3D-OMRI of a free radical injected in tumor-bearing mice can be performed at high anatomical resolution at a constant field. A 30 mm cavity operating at 5.43 GHz was inserted in a C-shaped magnet for proton MRI at 0.194 T. Nude mice with or without brain-implanted C6 rat glioma were positioned in the cavity and injected with TOPCA (1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole-3-carboxylic acid). OMRI was performed in 3D within several minutes in the brain region without high overheating of the animals. Voxel size was 0.5 × 0.5 × 1 mm³ , providing good delineation of brain regions. Signal amplifications ranged from 2 in tumors to 10 in vessels several minutes after TOPCA injection. Time-course of signal enhancement could be measured by 2D OMRI at 15 s time intervals in a localized thin slice. The method opens the way for molecular imaging of biological activities able to generate OMRI-visible free radicals., (Copyright © 2012 John Wiley & Sons, Ltd.)

A 9 GHz EPR imager that is capable of the imaging surface area of thin materials has been built. The EPR imager resolved samples spaced 1 mm apart. The developed TE(111) cavity was able to detect easily ~1.0 mM aqueous TEMPOL solution in ~1.0 mm (i.d.) glass capillary placed just above the cavity. The sensitivity measured using the TEMPOL solution showed ~0.3 of that for the modified JEOL cavity, which was in a qualitative agreement with the calculations considering the difference in the filling factors of the cavities. The relatively low measured sensitivity of the TE(111) cavity is due to utilization of the microwave field from ~3 mm aperture (hole) in the cavity wall. More importantly, the TE(111) cavity does not require inserting the sample into the cavity and placing samples into EPR tubes.

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., (Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.)

The structure of the hydrogen bond network is a key element for understanding water's thermodynamic and kinetic anomalies. While ambient water is strongly believed to be a uniform, continuous hydrogen-bonded liquid, there is growing consensus that supercooled water is better described in terms of distinct domains with either a low-density ice-like structure or a high-density disordered one. We evidenced two distinct rotational mobilities of probe molecules in interstitial supercooled water of polycrystalline ice [Banerjee D, et al. (2009) ESR evidence for 2 coexisting liquid phases in deeply supercooled bulk water. Proc Natl Acad Sci USA 106: 11448-11453]. Here we show that, by increasing the confinement of interstitial water, the mobility of probe molecules, surprisingly, increases. We argue that loose confinement allows the presence of ice-like regions in supercooled water, whereas a tighter confinement yields the suppression of this ordered fraction and leads to higher fluidity. Compelling evidence of the presence of ice-like regions is provided by the probe orientational entropy barrier which is set, through hydrogen bonding, by the configuration of the surrounding water molecules and yields a direct measure of the configurational entropy of the same. We find that, under loose confinement of supercooled water, the entropy barrier surmounted by the slower probe fraction exceeds that of equilibrium water by the melting entropy of ice, whereas no increase of the barrier is observed under stronger confinement. The lower limit of metastability of supercooled water is discussed.

This study deals with the identification of a title compound, 2,6-dimethyl-4-nitropyridine N-oxide by means of theoretical calculations. The optimized molecular structures, vibrational frequencies, corresponding vibrational assignments, thermodynamic properties and atomic charges of the title compound in the ground state were evaluated using density functional theory (DFT) with the standard B3LYP/6-311G(d,p) method and basis set combination for the first time. Theoretical vibrational spectra were interpreted with the aid of normal coordinate analysis based on scaled density functional force field. The results show that the optimized geometric parameters (bond lengths and bond angles) and vibrational frequencies were observed to be in good agreement with the available experimental results. Based on the results of comparison between experimental results and theoretical data, the chosen calculation level is powerful approach for understanding the molecular structures and vibrational spectra of the 2,6-dimethyl-4-nitropyridine N-oxide. Moreover, we not only simulated frontier molecular orbitals (FMO) and molecular electrostatic potential (MEP) but also determined the transition state and energy band gap. Based on the investigations, the title compound is found to be useful to bond metallically and interact intermolecularly. Infrared intensities and Raman activities were also reported., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

Despite the major impact of ROS on human health, their quantification remains difficult and requires an analytical approach, such as the EPR spin trap technique. In this study, a comparative EPR analysis of different macrophage types stimulated for superoxide and nitric oxide production was performed. U937 monocytes, J774A.1, RAW 264.7 and primary mouse (PMM) macrophages were included. In contrast to the U937 cells, all macrophages produced significant EPR signals after stimulation. The use of PMA as stimulator and CM-H as spin probe led to the highest response in EPR signals for detection of O(2)(.-) as nitroxide radical. A combination of LPS and IFN-gamma and the spin trap [Fe(DETC)(2)] turned out to be the best combination for the production and detection of intracellular NO spin adducts. In conclusion, this study established practical experimental conditions for the EPR analysis of O(2)(.-) and NO produced by different types of activated macrophages.

Electron paramagnetic resonance (EPR) in conjunction with a slow-tumbling simulation was utilized for defining stratum corneum (SC) lipid structure. We found that ordering calculated from the simulation is an appropriate index for evaluating SC lipids structure. The SC from two sites (mid-volar forearm and lower-leg) of human volunteers was stripped consecutively from one to three times using a glass plate coated with a cyanoacrylate resin. Aliphatic spin probes, 5-doxylstearic acid (5-DSA) and 3beta-doxyl-5alpha-cholestane (CHL), were used to monitor SC ordering. EPR spectrum of 5-DSA incorporated in the SC demonstrated a characteristic peak for the first strip. However, EPR spectra of CHL in the SC did not show a clear difference for each strip, except for the peak intensity. The results imply that CHL is not incorporated into the lipid phase as easily as 5-DSA is. A slow-tumbling simulation of the EPR spectrum was performed to analyze the detailed lipid structure. The simulation results for 5-DSA show differences in values of the SC ordering as a function of depth. Thus, these results along with the simulation analysis provide a detailed SC layer structure.

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.

Background/objectives: To measure reactive oxidant production and the decline in antioxidant potential in commercially available, irradiated parenteral nutrition (PN) solutions and the effect that these have on oxidant production in patients in the intensive care unit., Subjects and Methods: Vitamin E and malondialdehyde in irradiated and nonirradiated commercially available, PN solutions were measured. The PBN (alpha-phenyl-n-test-butylnitrone (PBN) spin trap was used to measure free radicals and TEMPOL (2,2,6,6-tetramethyl-4-hydroxy-piperidine-oxyl) was used to assess antioxidant capacity. The irradiated PN was administered (as per unit protocol) to 10 patients with gut failure and plasma and urinary isoprostanes and interleukin-6 (IL-6) were measured 1 hour preadministration, at the time of, and 1 and 2 hours postadministration of PN., Results: Irradiation reduced vitamin E significantly (P < .0025). Malondialdehyde products were present in both samples, but more so in irradiated samples (P < .0001), as were free radicals measured by PBN spin trapping. Irradiated samples had a higher scavenging capacity of TEMPOL free radical due to depletion of antioxidants in irradiated samples. Urinary isoprostanes increased at time 2 by 6.3 units relative to time 0 and by 5.23 units relative to time 1(Friedman ANOVA: P < .01413)., Conclusions: Lipid hydroperoxides are formed in PN solutions and increase further following irradiation. This is associated with a significant reduction in vitamin E and antioxidant potential. The increase in urinary isoprostanes indicates a potentially proinflammatory effect of irradiated PN.

Tetramic acid derivatives are an important class of nitrogen heterocycles with a pyrrolidine-2,4-dione core as a key structural motif. From the sponge-derived fungus Beauveria bassiana, a new equisetin-like tetramic acid derivative, beauversetin (1), was isolated. The sea weed-derived fungus Microdiplodia sp. produced the tetramic acid derivative 2 (Sch210972) which was shown to inhibit human leucocyte elastase (HLE) with an IC50 of 1.04 microg mL(-1).

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., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

Gynura segetum (Lour.) Merr. (Jusanqi) is a traditional herbal product used for hemostasis and detumescence in Chinese folk medicine. However, its hepatic toxicity should not be ignored. In this study, pyrrolizidine alkaloids (PAs) and their corresponding N-oxides (PANOs) were extracted from the whole plant of G. segetum and analyzed by high-performance liquid chromatography coupled to ion trap mass spectrometry (ITMS). Identification of eluted peaks as PANOs was indicated by virtue of their MS and MS(n) analysis, in addition to the [M+H](+) adduct ion, characteristically showed a significant (usually 100% abundance) dimer adduct [2M+H](+) that is not observed in the MS of the parent PAs. A total of 20 compounds were identified or tentatively characterized based on their mass spectra and possible biosynthetic pathways, of which three PAs and one PANO, namely seneciphylline, senecionine, seneciphylline and seneciphyllinine N-oxide, were unequivocally characterized, while other PAs and PANOs were tentatively assigned. Sixteen constituents were reported for the first time from G. segetum and tetrahydrosenecionine has not been previously reported as a natural product. Our results are the first comprehensive analysis of PAs and PANOs in G. segetum constituents and will be helpful for the quality control of the herb of G. segetum and its related preparations., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

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.

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.

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.

Aim: To investigate the potential of quorum sensing inhibitors (QSI) as food preservative agents in a food product, where bacterial spoilage is controlled by quorum sensing (QS)., Methods and Results: The effects of well-known QSI were tested on spoilage phenotypes and on QS-regulated genes of a bean sprout spoiling bacterial isolate (Pectobacterium A2JM) in laboratory substrates and in a bean sprout model system. The acylated homoserine lactones (AHL) analogues PenS-AHL and HepS-AHL decreased the specific protease activity of Pectobacterium A2JM in broth but did not reduce the expression of a QS-regulated secretion protein, and were without effect on soft rot of bean sprouts. The QSI ProS-AHL, furanone C-30, patulin, penicillic acid and 4-nitropyridine-N-oxide did not have any effect on protease activity, on gene expression or bean sprout appearance at nongrowth inhibitory concentrations. Extracts from garlic and bean sprouts induced the QS system of Pectobacterium in bean sprouts and a broth system, respectively., Conclusions: Among the several well-known QSI compounds, only PenS-AHL and HepS-AHL, inhibited QS-regulated protease activity of Pectobacterium A2JM in broth cultures, but had no effect on bean sprout spoilage., Significance and Impact of the Study: The QSI compounds must be selected in the specific system in which they are to function and they cannot easily be transferred from one QS system to another.

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.

Electron paramagnetic resonance spectroscopy is used to monitor the bioavailability of a nitroxide spin probe, 4-hydroxy-Tempo or Tempol, in Ca-hectorite suspensions and pastes, to bacteria capable of degrading this probe co-metabolically. In nutrient solutions with an initial probe concentration of 1.2 mM and in the absence of hectorite, bacteria are able to denature Tempol and eliminate its paramagnetic signal within 48 h. In the presence of hectorite and after flocculation, the effect of bacteria is significantly delayed, but almost complete denaturation still occurs, after roughly 120 h. When hectorite is added but the bacterial/clay suspension is not centrifuged, Tempol denaturation levels off after about 24 h and reaches a plateau with approximately 45% of Tempol remaining. This plateau does not constitute evidence of limited bioavailability, as is widely assumed, since subsequent addition of nutrients causes the denaturation reaction to proceed to a second plateau, with merely 10% of Tempol remaining.

We show that a Gaussian-shaped pulse can be used to excite selected 1H signals in hydroxyapatite, monetite and H-Y zeolite loaded with trimethylphosphine oxide (TMPO). This selective excitation method can be incorporated into Lee-Goldburg (LG) cross-polarization to obtain useful spectral editing opportunity. This new strategy has been applied to identify the Brønsted and the Lewis acid sites in H-Y zeolite using TMPO as the probe molecule.

The aim of the present study was to elucidate the beneficial effects of rosuvastatin, a new HMG-CoA reductase inhibitor, on colonic mucosal damage and on the inflammatory response in a dextran sulfate sodium (DSS) colitis model. Acute colitis was induced using 8% DSS in female BALB/c mice. Colonic mucosal inflammation was evaluated clinically, biochemically, and histologically. Mucosal protein contents and mRNA levels of tumor necrosis factor (TNF)-alpha were determined by immunoassay and real time-PCR. The mRNA levels of endothelial nitric oxide synthase (eNOS) were determined by real-time PCR. Disease activity scores in DSS-induced colitis model mice, as determined by weight loss, stool consistency, and blood in stool, were significantly lower in the rosuvastatin-treated mice than in control mice. Shortening of the colon was significantly reversed by rosuvastatin. Increases in tissue-associated myeloperoxidase activity and thiobarbituric acid-reactive substances after DSS administration were both significantly inhibited by treatment with rosuvastatin. Rosuvastatin also inhibited increases in intestinal TNF-alpha protein and mRNA expression after DSS administration, respectively. The mucosal mRNA levels of eNOS were decreased after DSS administration, but preserved in mice treated with rosuvastatin. These results suggest that rosuvastatin prevents the development of DSS-induced colitis in mice via the inhibition of mucosal inflammatory responses associated with the preservation of eNOS transcription.

A method has been developed to determine orientation distribution function (ODF) of anisotropic paramagnetic species by analysis of the angular dependence of the ESR spectra. The method is based on computational spectra simulation. The ODF is represented as an expansion in terms of orthonormal functions. The expansion coefficients are determined through minimization of discrepancies between simulated spectra and experimental ones. By means of the suggested method we have determined the orientation distribution functions for radical probe 2,2,6,6-tetramethyl-4-ol-piperidinooxyl in 4-n-amyl-4'-cyanobiphenyl aligned by magnetic field and 2-septadecyl-2,3,4,5,5-pentamethylimidazolidine in polyethylene stretched films. In each case, thermal evolution of the ODF has been investigated.

The reactions of benzo[h]quinoline N-oxide with methylsulfinyl carbanion and deuterated methylsulfinyl carbanion, respectively, were studied theoretically. Differences in yield between these reactions were explained using ab initio molecular orbital methods by considering the zero-point energy correction and the barrier penetration effect. In these reactions, two transition states affected the total reaction rate. The hydrogen- or deuterium-transfer step played a significant role, accounting for the difference in reaction rates.

5,5-Dimethyl-1-pyrroline N-oxide (DMPO) spin trapping in conjunction with antibodies specific for the DMPO nitrone epitope was used on hydrogen peroxide-treated sperm whale and horse heart myoglobins to determine the site of protein nitrone adduct formation. The present study demonstrates that the sperm whale myoglobin tyrosyl radical, formed by hydrogen peroxide-dependent self-peroxidation, can either react with another tyrosyl radical, resulting in a dityrosine cross-linkage, or react with the spin trap DMPO to form a diamagnetic nitrone adduct. The reaction of sperm whale myoglobin with equimolar hydrogen peroxide resulted in the formation of a myoglobin dimer detectable by electrophoresis/protein staining. Addition of DMPO resulted in the trapping of the globin radical, which was detected by Western blot. The location of this adduct was demonstrated to be at tyrosine-103 by MS/MS and site-specific mutagenicity. Interestingly, formation of the myoglobin dimer, which is known to be formed primarily by cross-linkage of tyrosine-151, was inhibited by the addition of DMPO.

The chrysomelid leaf beetles Longitarsus jacobaeae, Oreina cacaliae, and O. speciosissima sequester pyrrolizidine alkaloids from their asteracean host plants and store them as nontoxic N-oxides. Previous analyses showed that Longitarsus is able to N-oxidize protoxic tertiary PAs, but did not resolve in which form N-oxides are taken up. For Oreina, beetles seem able to directly transmit the polar PA N-oxides from the gut into the hemolymph and prevent any reduction of them in the gut yielding protoxic free bases. Here, we confirm the predicted direct uptake of PAs as N-oxides by Oreina, and elucidate the situation for Longitarsus by applying double-labeled [14C]senecionine [18O]N-oxide as tracer. The beetles were fed with the tracer and subsequently senecionine N-oxide was recovered from the defensive secretions (Oreina) and beetle extracts (Longitarsus), purified by HPLC, and submitted to ESI-MS, GC-MS, and analysis of the specific radioactivity. The 18O-label is retained without any loss in the labeled senecionine N-oxide recovered from the two Oreina species. Analysis of the Longitarsus experiment was complicated by a contamination of the HPLC-purified senecionine N-oxide with a second compound, identified as a dihydrosenecionine N-oxide by high-resolution CID analysis. The dihydrosenecionine N-oxide, probably the 15,20-dihydro derivative, constitutes a major idiosyncratic senecionine metabolite present in the beetle. The recovered senecionine N-oxide retained 74% 18O-label. The remaining 25% is mostly due to loss of 18O by reduction and subsequent re-N-oxidation. The experiments confirm for both beetle genera a direct uptake of the polar nontoxic PA N-oxides, which requires specific membrane carriers. Accumulation of detrimental free base PA is prevented by glucosylation (Oreina) or N-oxidation (Longitarsus).

Substituted benzyl alcohol was oxidized enzymatically with a laccase-mediator system and the products were investigated as a function of time by nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (nanoESI-FTICRMS). With Trametes versicolor laccase (TVL), the mediator, 2,2',6,6'-tetramethylpiperidine-N-oxyl radical (TEMPO), undergoes oxidation and forms oxoammonium ion. Oxidized TEMPO oxidizes the alcohol and is simultaneously reduced to the N-OH form. The laccase then restores TEMPO back to the normal radical form and the oxidation cycle starts again. The role of TEMPO and the structures of its oxidized and reduced forms in the enzymatic oxidation process were clarified in collision-induced dissociation experiments and gas-phase hydrogen/deuterium (H/D) exchange reactions. The amounts of enzyme and mediator were significant for product formation: with greater amounts overoxidation products, the corresponding benzoic acid and benzonitrile were formed. Smaller amounts of laccase and mediator generated benzaldehyde in high yield. The reaction pathway for benzonitrile formation is discussed and it is suggested to start from benzaldehyde and the ammonia in the ammonium acetate buffer., (2004 John Wiley & Sons, Ltd.)

Identification of reactive oxygen species (ROS) that contribute to bisphenol-A (BPA) degradation and monitoring of BPA at various concentrations in human serum under Fenton reaction conditions were carried out using electron spin resonance (ESR) spectrophotometry and high performance liquid chromatography with electrochemical detection (HPLC-ECD). BPA recovery decreased with increasing Fe concentration and time, both with a Fenton reaction using Fe(II), and with a Fenton-like reaction using Fe(III). In these reactions, BPA dose-dependently decreased the intensity of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-*OH, up to 1 microg/ml BPA, and no change in DMPO-O(2)(?-) intensity was observed. The decrease in BPA recovery was inhibited strongly by addition of serum under Fenton-like reaction conditions, and there was a negative correlation between turbidity and BPA recovery. To clarify the mechanism by which serum inhibits BPA degradation, the relationship between BPA recovery and sample turbidity, and characteristics of the precipitates were investigated using spectrophotometry and X-ray analysis. The precipitate formed in the serum-containing sample consisted of C, S, O, P and Fe. BPA degradation was also inhibited under Fenton-like reaction conditions in phosphate buffered saline (PBS), and a precipitate consisting of O, P, and Fe appeared. Precipitates also appeared in authentic albumin and gamma-globulin when sulfate was added with Fenton reagents. After precipitate removal, both Fe and protein concentrations in the supernatant of the protein solutions with sulfate decreased with increasing Fe addition. We demonstrate here that hydroxyl radical generation from Fenton or Fenton-like reactions can degrade BPA, and that serum strongly inhibits BPA degradation, not only by competing with BPA for hydroxyl radicals, but also by trapping Fe with oxidative components present in the serum.

Cellulose cotton linter was oxidized with sodium hypochlorite with catalytic amounts of sodium bromide and 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) under various conditions. After this TEMPO-mediated oxidation, water-insoluble fractions were collected and characterized in terms of carboxylate and aldehyde contents, crystallinities and crystal sizes, degrees of polymerization, morphology, and water retention values. Carboxylate and aldehyde groups were introduced into the water-insoluble fractions up to about 0.7 and 0.3 mmol/g, respectively, by the oxidation, where recovery of the water-insoluble fractions were generally higher than 80%. Crystallinities and crystal sizes of cellulose I were nearly unchanged during the oxidation, and thus, carboxylate and aldehyde groups were introduced selectively on crystal surfaces and in disordered regions of the water-insoluble fractions. Water retention values of cotton linter can be increased from 60% to about 280% through the introduction of hydrophilic carboxylate groups and morphological changes from fibrous forms to short fragments by the TEMPO-mediated oxidation.

In this paper, we propose a novel approach for electron paramagnetic resonance (EPR) mixture spectra analysis based on blind source separation (BSS) technique. EPR spectrum of a free radical is often superimposed by overlapping spectra of other species. It is important and challenging to accurately identify and quantify the 'pure' spectra from such mixtures. In this study, an automated BSS method implementing independent component analysis is used to extract the components from mixed EPR spectra that contain overlapping components of different paramagnetic centers. To apply this method, there is no requirement to know the component spectra or the number of components in advance. The method is applied to analyze free radical EPR spectra which are collected from standard chemical system, cultured cell suspense, and ex vivo rat kidneys by spin trapping EPR technique. Results show that the BSS method proposed here is capable of identifying the component EPR spectra from mixtures with unknown compositions. The BSS technique can offer powerful aids in resolving spectral overlapping problems in general EPR spectroscopy analysis.

The kinetics of the reaction between superoxide and the spin trapping agents 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), and 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) were re-examined in the superoxide-generating xanthine/xanthine oxidase system, by competition with spontaneous dismutation. The approach used singular value decomposition (SVD), multiple linear regression, and spectral simulation. The experiments were carried out using a two-syringe mixing arrangement with fast scan acquisition of 100 consecutive EPR spectra. Using SVD analysis, the extraction of both temporal and spectral information could be obtained from in a single run. The superoxide spin adduct was the exclusive EPR active species in the case of DEPMPO and BMPO, and the major component when DMPO was used. In the latter case a very low concentration of hydroxyl adduct was also observed, which did not change during the decay of the DMPO-superoxide adduct. This indicates that the hydroxyl radical adduct is not formed from the spontaneous decay of the superoxide radical adduct, as has been previously suggested [correction]. It was established that in short-term studies (up to 100 s) DMPO was the superior spin trapping agent, but for reaction times longer than 100 s the other two spin traps were more advantageous. The second order rate constants for the spin trapping reaction were found to be DMPO (2.4 M(-1)s(-1)), DEPMPO (0.53 M(-1)s(-1)), and BMPO (0.24 M(-1)s(-1)) determined through competition with spontaneous dismutation of superoxide, at pH 7.4 and 20 degrees C.