ArticleLiterature Review

Electron Spin Resonance.: Part One: A Diagnostic Method in the Biomedical Sciences

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  • Fresh-lands Environmental Actions, Caversham, United Kingdom.
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Abstract

A review is presented of some of the ways in which electron spin resonance (ESR) spectroscopy may be useful to investigate systems of relevance to the biomedical sciences. Specifically considered are: spin-trapping in biological media; the determination of antioxidant efficiencies; lipid-peroxidation; the use of nitroxides as probes of metabolic activity in cells and as structumral probes of cell-membranes; ESR coupled with materials for radiation-dosimetry; food- and drug-irradiation; studies of enzyme systems and ofcyclodextrins; diagnosis of cancer and rheumatoid arthritis; measurement of oxidative stress in synovial tissue in preparation for joint replacement; determination of oxidative species during kidney dialysis; measurement of biological oxygen concentrations (oximetry); trapping in living cells of the endothelium-derived relaxing factor nitric oxide (NO); measurement of hydrogen peroxide; determination of drugs of abuse (opiates); ESR measurements of whole blood and as a means to determine the age of bloodstains for forensic analysis are surveyed, and also a determination of the aqueous volume of human sperm cells is described, among other topics.

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... ROS and RNS possess a short life time and thus are difficult to measure. Electron spin resonance (ESR)-also known as electron paramagnetic resonance (EPR), is a suitable method to measure these unstable molecules, 16 and EPR allows quantification of various free radical species. 17 There are two types of spin traps, nitrone and nitroso compounds. ...
... Trapping methods are useful in vitro and in animal studies, but their usefulness in human studies have been limited, and unfortunately these are a very expensive techniques due to the special and pricy equipment required. 16 ...
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... The vast growing of proteomics, genomics and the sequences of protein generated a significant gap between the number of known structure and the sequence-known of proteins. Constraints for example cost and time of methods for protein structure determination such as (X-ray [4], NMR [5] and ESR [6]) Which means that they may not be sufficient to resolve each structural sequence of the protein. Thus, the development of a reliable computation method for determine the class of protein is very demanding. ...
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... The exponential growth of genomics, proteomics and protein sequences created a large gap between the number of sequence-known and structure-known proteins. Limitations such as time and costs of the common techniques for determining protein structures X-ray crystallography (Rupp, 2009), nuclear magnetic resonance (NMR) (Press, 2007), electron spin resonance (ESR) (Rhodes, 2011) suggest that they might not be adequate for solving all protein structural sequence. Hence, developing a reliable computational method for determining the protein structural classes is quite challenging. ...
... Electron paramagnetic resonance spectroscopy is based on the resonant absorption of microwave radiation by matching the energy difference of the spins of free unpaired electron in a magnetic field, and spin reversal and absorption of microwave energy can be measured [73]. The rotational effect in tissue water with a significant damping induced by the resonant absorption of microwave radiation should be considered to avoid a consequence of the high impedance at this frequency level (10 9 Hz). ...
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... Recent developments in EPR are described in a review series . Biomedical applications of EPR of radicals are outside the scope of this review, but are reviewed by Rhodes (Rhodes 2011). ...
Chapter
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A description of 2 ready to use pre-compiled programs for the low-resolution estimation of molecular shape from hydrodynamic measurements, and the prediction of the non-ideality parameter known as the 2nd virial coefficient from shape information
... Recent developments in EPR are described in a review series . Biomedical applications of EPR of radicals are outside the scope of this review, but are reviewed by Rhodes (Rhodes 2011). ...
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... The used method of visible wavelength range by SPD applied in this study is enough confidential and simple for laboratory use for aforementioned reactant ABTS in comparison to others. In addition, many alternative methods exist for similar investigation on the antioxidativity potential such as electron-spin resonance [19,20]. Chemiluminescence is based on the recording energy released during chemical reaction through the light emission [21]. ...
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... However, few antioxidants including edaravone (to treat ischaemic stroke in Japan) have found accepted clinical use. Moreover, many well-known substances, including antioxidant vitamins (A, C and E), and more recently developed materials like nitrones (also used as spin-traps for radicals in electron spin resonance investigations 16 ), have not unanimously passed the scrutiny of clinical trials that they are effective in the prevention and treatment of various diseases. To date, there have been several large (47,000 participants) clinical trials aimed to test the effectiveness of antioxidants as cancer prevention agents specifically, none of which have been convincing 17 . ...
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Chapter
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Chapter
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The ortho-quinodimethane 1 reacts quickly and efficiently as a diradical equivalent in a cheletropic reaction with the radical .NO to form 2, which can be very easily observed through ESR spectroscopy. The detection by this technique of the formation of 2 from 1 and .NO released by liver macrophages demonstrates that diradical equivalents of type 1 could be suitable for monitoring .NO production under physiological conditions.
Article
Pure nitric oxide does not add to alkenes containing acceptor or donor substituents, or to conjugated dienes, to afford β-nitrosoalkyl radicals. EPR spectra show that reactions are initiated by NO2 addition to carbon–carbon double bonds to produce β-nitroalkyl radicals which combine with nitric oxide to yield β-nitro-nitroso-compounds. The latter trap other radicals to afford mixtures of aminoxyl radicals and, where possible, the nitroso-compounds tautomerise to oximes which oxidise to iminoxyl radicals. EPR spectra obtained on reaction of NO–NO2 mixtures with 2,5-dimethylhexa-2,4-diene have shown the presence of a di-tert-alkylaminoxyl plus a tert,sec-dialkylaminoxyl, providing good support for this mechanism. Product analyses have established that 2-oxo-4-methylpent-3-ene and 1-acetylcyclohexene undergo extensive oxidative degradation. For enol ethers, reaction with NO–NO2 leads to hydrolysis giving the corresponding carbonyl compound and alcohol, followed by ketal and ester formation.
Article
Two isomers of permethylated β-cyclodextrin, labelled with paramagnetic TEMPO moieties attached on the same rim of the cavity, were synthesised and their host–guest properties investigated by EPR spectroscopy.
Article
Addition of ˙NO to dilute (ca. 10–4 mol l–1) solutions of 2,3-dimethylbuta-1,3-diene gave at least two nitroxyl radicals. The major radical, with a half-life of ca. 30 min, had an EPR spectrum with 1H hyperfine splitting of ca. 10 G from four equivalent protons. Similarly, the major nitroxyl radical formed from 2,5-dimethylhexa-2,4-diene gave a major triplet showing no resolved 1H hyperfine coupling, but satellite features from 13C (a= 10 G) from two equivalent nuclei. A minor radical with slightly different 14N coupling and a 13C splitting of 6.5 G was also detected. These radicals were very stable. On dilution of the diene, the second species was favoured over the former, and became the major component at 10–5 mol l–1. These reactions occurred in the complete absence of ˙NO2. No radicals were detected using pure ˙NO2, but mixtures gave strong signals from nitroxyl radicals.
Article
Nω-Hydroxy-L-arginine (2) was prepared by a multi-stage synthesis; the key step was the addition of hydroxylamine to the protected cyanamide 8. The presence of N-hydroxyguanidines was confirmed, above all, by 15N-NMR investigations. 15Nω-Hydroxy-L-arginine (2) was converted quantitatively to 15NO by NO synthases from macrophages. 15NO was identified by ESR-spectroscopy. These experiments confirm that 15Nω-hydroxy-L-arginine (2) is an intermediate in the biosynthesis of NO from arginine (1) and that the N-hydroxylated N-atom is present in the NO formed. Synthese, ESR- und 15N-NMR-Untersuchungen von 15Nω-Hydroxy-L-arginin zur Aufklärung der enzymatischen Stickstoffmonoxidbildung aus L-Arginin Nω-Hydroxy-L-arginin (2) wurde in einer mehrstufigen Synthese gewonnen, wobei der entscheidende Schritt die Addition von Hydroxylamin an das geschützte Cyanamid 8 darstellt. Das Vorliegen von N-Hydroxyguanidinen wurde besonders durch 15N-NMR-Studien bestätigt. 15Nω-Hydroxy-L-arginin (2) wurde von NO-Synthase aus Makrophagen quantitativ zu 15NO umgesetzt. 15NO wurde ESR-spektroskopisch nachgewiesen. Die Studien belegen, daß Nω-Hydroxy-L-arginin (2) eine Zwischenstufe bei der Biosynthese von NO aus Arginin (1) darstellt und ausschließlich der N-hydroxylierte Stickstoff im gebildeten NO erscheint.
Article
The selenite radical anion, SeO3.-, can be detected by ESR spectroscopy using water-soluble spin-traps, N-(4-pyridylmethylene)-t-butylamine N,N'-dioxide (POBN) and 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS), but selenate radical anion, SeO4.-, cannot be trapped by POBN or DBNBS.
Article
A model previously developed for describing the dynamics of flexible alkyl chains that is based on Flory's rotational isomeric state approximation is adapted and applied to the analysis of electron spin resonance (ESR) spectra obtained from a phospholipid spin label in a macroscopically aligned phospholipid membrane. In this model, rotation around each C−C bond of the labeled alkyl chain is characterized by three inequivalent minima, with one end of the chain fixed to mimic the phospholipid headgroup, and with the dynamic effects of the nitroxide label explicitly included. This model is integrated with that for the overall rotation of the phospholipid in the mean orientational potential of the aligned membrane, and it is incorporated into the stochastic Liouville equation which describes the ESR line shape in the presence of these dynamic processes. The analysis is simplified by introducing the fact that the relatively rapid internal modes of motion can be treated by motional narrowing theory and a time scale separation can be made with respect to the much slower overall motions of the phospholipid. A series of ESR spectra from the spin label 16-PC in the lipid dimyristoylphosphatidylcholine were obtained over a range of temperatures (35−65 °C) in the Lα phase for various orientations of the normal to the bilayer plane relative to the magnetic field. Very good agreement with experiment is obtained from this model by using least squares fitting procedures for the overall motional dynamics. One finds an order parameter of that is constant throughout the phase and the perpendicular component for rotational diffusion, R, that ranges from about 1−3 × 107s-1 (which corresponds to the ESR slow motional regime). Fits to the ESR spectra were also obtained from a simple but standard model wherein a single overall rotational diffusion tensor is used to describe the combined effects of internal and overall dynamics. These fits were almost as good, but they lead to a much larger R ≈ 3−6 × 108 s-1 and a smaller = 0.1, since these parameters now include the composite effects of both types of processes. New ESR experiments are proposed to provide more critical tests of these models.
Article
Two-dimensional Fourier transform (2D-FT) and CW-ESR experiments at X-band frequencies were performed over a broad range of temperatures covering the solid and melt states of a liquid crystalline (LC) polymer. The CW-ESR experiments were analyzed by conventional motional models. The nematic phase was macroscopically aligned in the dc magnetic field, whereas the solid state showed microscopic order but macroscopic disorder (MOMD). An end-label on the polymer showed smaller ordering and larger reorientational rates than that of the cholestane (CSL) spin probe dissolved in the same polymer, since the former can reorient by local internal chain modes. It was demonstrated that the 2D-FT-ESR experiments provide greatly enhanced resolution to the ordering and dynamics of the end-label, especially when performed as 2D-ELDOR (electron−electron double resonance) experiments as a function of mixing time. The conventional model of Brownian reorientation in an orienting potential was unsuccessful in interpreting these results. Instead the model of a slowly relaxing local structure (SRLS), which enables differentiation between the local internal modes experienced by the end-label and the collective reorganization of the polymer molecules around the end label, yielded much improved fits to the experiments in the nematic phase. These nonlinear least squares fits showed that the polymer “cage” relaxes more than 2 orders of magnitude slower than the local end-chain modes, and there is a moderate orientational potential coupling the local end-chain motion to the “cage” with axial and nonaxial local order parameters of about 0.14 and 0.29 at 100 °C. In the solid state the 2D-ELDOR spectra were fitted to the MOMD model, which is a limiting case of the SRLS model when the cage relaxation becomes very slow.
Article
Precursors for the selective generation of C-2 glyceryl radicals were synthesized, and the chemical behavior of the corresponding radicals was investigated by ESR spectroscopy, product analysis, and kinetic measurements. It was found that cleavage of the β-C,O bond proceeds rapidly, if a hydroxyl group is present at the radical carbon center. The rate constant for the elimination of a β-acetoxy group from radical 30 was dependent on the solvent (kE = 4 × 105 s-1 in methanol, kE = 2 × 107 s-1 in toluene). With these results and ab initio calculations a concerted elimination mechanism is suggested. α-Methoxy-substituted C-2 glyceryl radicals 42 and 43 showed heterolytic β-C,O bond cleavage under formation of radical cations. With ester-substituted radicals 24 and 35 no elimination could be observed. To demonstrate the biological significance of these findings, C-2 lysolecithin radical 53 was generated, which led to fast β-elimination.
Article
This work reports an investigation of the free-radical chemistry of cholesterol and a number of cholesterol derivatives and analogues. The initial radicals formed in solid oxygen-free cholesterol samples after γ-irradiation at 77 K are found to be a tertiary side-chain radical and an allylic radical in the cholesterol A and B rings. At 300 K only the allylic radical is found. The structure of the allylic radical is confirmed by experiments on two analogues of cholesterol, 7-OH cholesterol, and the selectively deuterated 7-D-7-OH cholesterol, both of which produce the allylic radical after γ-irradiation by loss of the hydroxyl group. Cholesterol samples with oxygen present provide evidence for formation of two distinct peroxy radicals originating with the two carbon radicals found in the oxygen-free samples. These peroxy radicals are suggested to have different reactivities resulting from the different motional freedom each possesses. We suggest that the products found after radiation-induced autoxidation at or near room temperature are consistent with the different reactivities of the peroxy radicals. It is found for four cholesterol esters, one cholesterol derivative, and one sterol that the only radical stable at room temperatures in oxygen-free samples is the allylic radical. In only one ester, cholesterol chloroacetate, is the allylic radical not the stable room temperature radical.
Article
The reactions of lipid carbon-centered radicals and their corresponding peroxyl radicals (ROâ{sup {sm bullet}}, LOâ{sup {sm bullet}}) with five phenolic antioxidants (BHT, BHA, PG, TBHQ, and vitamin E) in frozen saturated and unsaturated lipids at low temperatures have been elucidated through electron spin resonance spectroscopy. The lipid radicals are formed after the γ or UV irradiation of the lipid matrices in the absence or presence of oxygen at 77 K. Upon warming oxygenated samples to 135 K, molecular oxygen adds to the carbon radicals forming peroxyl radicals. At 170 K peroxyl radicals in tributyrin react with added antioxidants (AH) to form antioxidant radicals (A{sup {sm bullet}}). The relative rates of reactions of these five antioxidants with the peroxyl radical of tributyrin decrease in the order BHT > TBHQ > E ⥠PG > BHA. For the reactions with triolein and trilinolein peroxyl radicals, the order of reactivity is BHT > E > BHA > TBHQ. This difference in reaction order of antioxidants in tributyrin and unsaturated lipids is explained by a dispersion model of antioxidants in lipid matrices.
Article
The kinetics of the low-temperature autoxidation of triglycerides has been investigated by electron spin resonance spectroscopy. After initial radical production, four reaction stages are found in the overall autoxidation of unsaturated lipids: (1) formation of peroxyl radicals by addition of molecular oxygen to the initial carbon radicals, (2) consumption of oxygen in the autoxidation cycle, (3) decay of the lipid peroxyl radical into allylic and pentadienyl radicals, and (4) recombination of the carbon-centered radicals. Peroxyl radical decay in saturated lipids follows second-order kinetics with an apparent activation energy of ca. 50 kJ/mol. The authors find that, for polyunsaturated lipids, even at quite low temperatures (120 K), the autoxidation process occurs readily and must be considered in the storage of biological samples.
Article
An electron spin resonance (ESR) study of the low-temperature autooxidation of lipids is reported in this work. Free radicals were generated at 77 K in the unsaturated lipids by means of UV photolysis of the lipid hydroperoxides normally found in these materials. For saturated lipids radicals were initiated either by photolysis of samples containing a small amount of added peroxide or by ..gamma..-irradiation. The initial radicals formed by photolysis (RO, OH) rapidly abstract from the lipid to form carbon-centered radicals. Annealing samples to temperatures which allow for molecular oxygen migration results in the formation of peroxyl radicals. The temperature at which oxygen migration starts is dependent on the lipid. For unsaturated lipids loss of LOâ with further annealing resulted in a concomitant production of the lipids allylic or pentadienyl radicals. In experiments with linolenic acid reintroduction of oxygen then results in formation of the lipid peroxyl radicals at low temperatures and the continued observation of the pentadienyl radical at higher temperatures. This is explained in terms of relative rates of oxygen migration and abstraction reactions. In the case of saturated lipids no new radicals appear with the loss of the peroxyl radicals. These results are discussed in light of previous work.
Article
The possibilities of investigations of the nanostructure of matter by pulsed EPR spectroscopy based on the measurement of the dipole–dipole coupling between the spins of unpaired electrons are considered. Using these methods, one can determine the conformations of long-chain organic biradicals and biomolecules (either doubly spin-labelled or containing paramagnetic metal ions) and study the supramolecular structure of matter, i.e., peptide clustering, heterogeneity of polymer packing, structural details of photosynthetic reaction centres and other complex systems. Investigations of the photoinduced spin-correlated radical pairs also reveal the regularities of charge transfer in these systems. The methods are characterised by high accuracy of measurements (0.03 nm) and can be used for measuring distances in the 1.5–10 nm range.
Article
A method to assess anti-oxidant activity quantitatively is presented. In this method, free radicals (OH·) were produced from the Fenton reaction. Electron spin resonance spectroscopy (ESR) was then used to determine the effectiveness of five anti-oxidants to scavenge the free radicals. Anti-oxidant activity was assessed as the percentage reduction of the ESR signal intensity relative to that of a control after 10 min. The order of the potency of the antioxidants at 4.8 mM was: caffeic acid > o-coumarin > 6,7-dihydroxy-4-methylcoumarin > catechin > scopoletin. In addition, pro-oxidant activity (a higher ESR signal intensity than that of the control) was observed for catechin and scopoletin at low concentrations (below 3.6 mM).© 2000 Society of Chemical Industry
Article
A range of drugs in the form of microcrystalline powder was exposed to γ-radiation. EPR measurements proved that all of them contained various paramagnetic species after 4 and 8 weeks of storage. We observed following radical concentrations, stable up to 4 weeks: the highest for ifosfamide — 4.5×1017 spins per gram and the lowest for nimodipine — 2.1×1016 spins per gram. Three drugs exhibited very weak EPR signals before irradiation, not detectable quantitatively. Some spectroscopic properties and suggestions concerning possible structure of the radicals are given in our paper.
Article
Positional displacements of specific groups on lipophilic molecules in membranes with respect to the lipid bilayer surface were used to describe the reduction of doxylmethyl palmitates by ascorbate in liposomes and erythrocytes. These highly lipophilic molecules are predominantly dissolved in the lipid bilayers of liposome and erythrocyte dispersions. Therefore the reaction rates reflect the probability of finding the nitroxides within the outer polar region of the membrane, which is accessible by ascorbate. A decrease of electron paramagnetic resonance intensity of a series of methyl esters of doxylpalmitic acids with different positions of nitroxides along the acyl chain was studied during the kinetics of ascorbate reduction. Models were introduced to evaluate the amplitudes of longitudinal positional motions of the nitroxides with respect to their equilibrium position within the membrane.
Article
One-dimensional electron paramagnetic resonance imaging (1D-EPRI) and EPR reduction kinetics were used to follow continuously the transport of liposome entrapped substances into the skin. Through 1D-EPRI the concentration distribution of the paramagnetic probe, which was applied to the skin entrapped in liposomes, could be followed, while through EPR reduction kinetics the chemical transformation of the paramagnetic probe, after it had been released from the liposomes, to an EPR-invisible form could be measured. Through the combination of both methods, and with the application of a model, in which the heterogeneity of different skin layers and the metabolism of the released substance was taken into account, liposome decay in the skin, as well as the time evolution of concentration distribution profiles for ASL in skin, was followed separately for both the entrapped substance and that released from liposomes. MLV (multilamellar vesicles) and REV (reverse-phase evaporation vesicles) obtained from egg lecithin and cholesterol (7:3 mol/mol) with the entrapped spin probe ASL N-(1-oxyl-2,2,6, 6-tetramethyl-4-piperidinyl)-N-dimethyl-N-hydroxyethylammonium iodide), which does not penetrate the liposome membrane easily, were applied to pig ear skin and the results were compared with those obtained for ASL dissolved in water and applied to the skin. The rapid decay of liposomes in the stratum corneum was measured, being much faster for MLV than for REV. In addition, a rate of transport 100-times faster was observed for ASL applied to the skin in REV than that observed for ASL applied in MLV or in solution. Our observations show that the rapid decay of liposomes takes place in the stratum corneum, however, some of the ASL molecules remain protected from the reducing agents in the skin, which indicates that some REV liposomes can penetrate deeper into the skin, or at least their lipids protect the entrapped substance from metabolic transformation.
Article
The stoichiometries of lipid-protein interaction obtained from spin label electron spin resonance experiments with integral membrane proteins are compared with simple geometric models for the intramembranous perimeter that are based on the predicted numbers of transmembrane helices. Deviations from the predicted values provide evidence for oligomerization of the protein in the membrane and/or more complex arrangements of the transmembrane segments.
Article
Guest-host complexes of β- and γ-cyclodextrins (CDs) with two spin-labeled indole derivatives having the same molecular weights but different structures were studied by EPR spectroscopy in aqueous solutions and semiempirical quantum-chemical calculations of these systems were carried out. In the presence of CD the polarity of the NO group environment decreases and the rotational correlation time (τ) of guest molecules increases. Both indole derivatives form 1 : 1 complexes with γ-CD, the binding constants of the complexes being different more than twice. Simulation of EPR spectra made it possible to determine the indole ring orientation relative to the plane of the host molecule (at angles in the range 30–60°) and the rotational diffusion coefficients of the complexes, which corresponded to the hydrodynamic volume of one γ-CD molecule. In contrast to the complexes with γ-CD the rotational correlation times, τ, of the complexes with β-CD correspond to a hydrodynamic volume which much exceeds the volume of a single β-CD molecule. The complexes with β-CD are also characterized by more hydrophobic environment for guest molecules and absence of spin exchange with Ni2+ ions in the aqueous solution. There results are consistent with a dimeric structure of β-CD in the complex and with the orientation of the long axis of the guest molecule along the dimer axis. The energies and geometric parameters were calculated for all complexes by the PM3 method with a conventional set of parameters. The optimized energetically stable structures of the 1 : 1 complexes with γ-CD and of the 1 : 2 complexes with β-CD are consistent with experimental data.
Article
This paper describes the rationale for carrying out EPR studies in human subjects in the clinical setting and the potential approaches and specific steps needed to make such studies feasible and useful. The suggested operational approach is to have the initial applications occur in as clinically useful and simple a manner as possible, with the expectation that once the technique is introduced and accepted in the clinical setting, that more complex and/or more technically difficult applications will be able to be developed. The initial approach should be based on EPR spectroscopy at 1.2 GHz focusing on clinical applications for which in vivo EPR provides a clearly useful approach to important clinical problems for which currently there is no good alternative approach, that can be carried out by measurements within 10 mm of the surface. The suggested initial clinical applications are: guiding tumor therapy for tumors and vascular disease by direct measurements of tissue pO2, characterizing and monitoring implanted drug delivery systems, and monitoring critical care.
Article
This item deals with identification of irradiated foodstuffs by means of Electron Paramagnetic Resonance (EPR). EPR is the most accurate method for such routine applications since radicals are stabilized for a long time in all (part of) foods which are in solid and dry state; consequently, EPR can be applied to meat and fish bones, fruit and relative products (from vegetal origin), seafoods, etc.
Article
Active carbons have been prepared from sucrose, using chemical activating agents (KOH and ZnCl2) to yield carbons with BET surface areas of 430–1170 m2 g−1, which are dependent on the amount of activating agent present during carbonization. Adsorption studies with nitrogen at 77 K have demonstrated type I adsorption/desorption isotherms with well-defined plateaus and no hysteresis. The adsorbent pore volume determined by the BJH technique is mostly microporous, with pore radii smaller than 20 Å, the degree of microporosity being dependent on preparation conditions. Additional characterization has been performed by monitoring the electron paramagnetic resonance (EPR) linewidth ΔBpp of the active carbons in the presence of various pressures of oxygen. The carbons give a single resonance line at g≈2.00, the free electron value. Subsequent analysis of the EPR characterization has shown a reversible dependence of the linewidth on the pressure of oxygen, Po2, and a dependence of the response d(ΔBpp)/dPo2 on the BET surface area. Thus active carbon oxygen sensors with differing responses to oxygen can be produced for oximetry applications.
Article
α-(4-Pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) radical adducts, which are formed in the reactions of soybean lipoxygenase with linoleic acid, arachidonic acid, and linolenic acid, were isolated using HPLC-ESR spectroscopy. Both linoleic acid and arachidonic acid gave one radical adduct, whereas in the case of linolenic acid, two radical adducts were isolated. These radical adducts all showed virtually identical uv spectra with λmax at 292 and 220 nm in hexane. The absence of absorbance with λmax at 234 nm indicates that a conjugated diene structure is not contained in these radical adducts. The mass spectra of the radical adducts formed from linoleic and arachidonic acids were identical and contained a molecular ion of 264, consistent with the trapping of the pentyl radical by 4-POBN. Indeed, authentic 4POBN pentyl radical adduct obtained from the reaction between pentylhydrazine and 4-POBN gave the same mass spectrum as the product obtained from the reaction of linoleic acid and arachidonic acid with 4-POBN. The two 4-POBN radical adducts formed in the linolenic acid reaction were shown by mass spectrometry to be isomers of pentenyl radicals. The 4-POBN-pentyl radical adduct was also detected in the reaction mixture of 13-hydroperoxy-linoleic acid, soybean lipoxygenase, and 4-POBN, indicating that the pentyl radical and pentenyl radical are formed by the decomposition of the hydroperoxides.
Article
A series of nitroxides was tested for rates of one-electron reduction in a chemical, a photochemical, and two biological systems by ESR assays. In all cases, piperidine and hydropyridine nitroxides were reduced consistently more rapidly than pyrroline and pyrrolidine nitroxides. Substituents on the nitroxides also affected reduction rates, although not as greatly as ring structure. One of the reduction systems, consisting of the photosensitizer FMN and the photoreductant EDTA, was used to study both anaerobic reduction and O2-dependent reoxidation of some of the nitroxides. Reduced piperidine and hydropyridine nitroxides were also oxidized more rapidly than the reduced pyrroline and pyrrolidine nitroxides. Reoxidation subsequent to reduction was partially inhibited by Superoxide dismutase, indicating that Superoxide radicals are involved in the process. Even after prolonged reoxidation, not all of the probe molecules were returned to their oxidized form, implying an irreversible “destruction” of the spin probe concomitant with its chemical reduction. Probe destruction was studied more specifically with a photochemical system for generating methyl radicals, which showed that these carboncentered radicals destroyed different nitroxides at rates which were much less influenced by the nitroxide structures than one-electron reduction was.
Article
One-dimensional electron paramagnetic resonance imaging (1D-EPRI) was used to evaluate quantitatively the influence of the size and composition of liposomes on the translational mobility of liposome-entrapped charged hydrophilic molecules (ASL) into the skin. It was found that only ASL molecules entrapped in small unilamellar vesicles (SUV) or reverse-phase evaporation vesicles (REV) from ‘fluid’ phospholipids (egg or soya lecithin and phosphatidylserine) can diffuse, while free ASL or ASL entrapped in the multilamellar vesicles (MLV) or REV from ‘solid’ phospholipids (dipalmitoyl lecithin/dipalmitoylglycerol) do not penetrate. The 1D-EPRI method was found to be useful for the study of diffusion of drugs into the skin.
Article
After a brief description of food irradiation treatments, the main methods for the identification of irradiated foodstuffs are described. A distinction is made between screening and proof methods, with emphasis on the main methods which have recently been published as official European protocols.
Article
ESR provides an excellent method for the identification of irradiated foods containing bone or calcified cuticle, even in the absence of unirradiated controls. It also shows promise for strawberries.