Spin trapping: an essential tool for the study of diseases caused by oxidative stress.

Electron spin resonance (ESR), called also electron paramagnetic resonance (EPR) together with the spin trapping technique, has allowed us to study and understand how free radicals are involved in various pathologies. In this review, the importance of spin trapping technique in the study of diseases such as cancer, diabetes, hypertension and parasitic diseases is discussed. In addition, advances in the use of this technique as therapeutic agents and other interesting applications as the immuno-spin trapping technique are reviewed.

Trapping of fatty acid allyl radicals generated in lipoxygenase reactions in biological fluids by nitroxyl radical.

Nitroxyl radicals can trap fatty acid allyl radicals on ferric-lipoxygenases at lower oxygen content, which are an intermediate in the lipoxygenase reaction. In the present study, we examined whether nitroxyl radical-trapping of fatty acid allyl radicals on the enzyme proceeds in biological fluids with abundant antioxidants. The fatty acid allyl radical-nitroxyl radical adducts were quantified by HPLC with electrochemical detection (HPLC-ECD); the adducts in eluate degraded into nitroxyl radical by passing through heating coil at 100 degrees C, and then nitroxyl radical was detected by electrochemical detector. Soybean 15-lipoxygenase and nitroxyl radical (3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl, CmDeltaP) were mixed with rat serum prepared from fresh venous blood, and the solution was stood at 37 degrees C for 1 h. One volume of the solution was mixed with 5 vols of cold acetonitrile. After centrifugation, the supernatant was subjected to HPLC-ECD. Arachidonate allyl radical-CmDeltaP adducts as well as linoleate allyl radical-CmDeltaP adducts were detected in the solution, and the content of these adducts remarkably increased in the presence of phospholipase A(2). It is proved for the first time that nitroxyl radical traps fatty acid allyl radicals generated in the lipoxygenase reaction in biological fluid without competition from endogenous antioxidants.

Immuno-spin trapping of protein and DNA radicals: "tagging" free radicals to locate and understand the redox process.

Biomolecule-centered radicals are intermediate species produced during both reversible (redox modulation) and irreversible (oxidative stress) oxidative modification of biomolecules. These oxidative processes must be studied in situ and in real time to understand the molecular mechanism of cell adaptation or death in response to changes in the extracellular environment. In this regard, we have developed and validated immuno-spin trapping to tag the redox process, tracing the oxidatively generated modification of biomolecules, in situ and in real time, by detecting protein- and DNA-centered radicals. The purpose of this methods article is to introduce and update the basic methods and applications of immuno-spin trapping for the study of redox biochemistry in oxidative stress and redox regulation. We describe in detail the production, detection, and location of protein and DNA radicals in biochemical systems, cells, and tissues, and in the whole animal as well, by using immuno-spin trapping with the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide.

Spin trapping experiments with different carbamoyl-substituted EMPO derivatives.

The spin trapping behavior of five carbamoyl-substituted EMPO derivatives, 5-aminocarbonyl-5-methyl-pyrroline N-oxide (CAMPO (AMPO)), 5-aminocarbonyl-5-ethyl-pyrroline N-oxide (CAEPO), 5-aminocarbonyl-5-propyl-pyrroline N-oxide (CAPPO), 5-aminocarbonyl-5-n-butyl-pyrroline N-oxide (CABPO), and 5-aminocarbonyl-5-n-pentyl-pyrroline N-oxide (CAPtPO), toward different oxygen- and carbon-centered radicals is described, the stabilities of the superoxide adducts ranging from about 8 to 17min.

Methods for nitric oxide detection during plant-pathogen interactions.

Nitric oxide (NO) is involved in the transduction of numerous signals in living organisms, and its biological effects are often influenced by its concentration. Therefore, the ability to reliably detect and quantify NO is crucial to understanding its role in cellular processes. Many techniques are available to detect and quantify NO, but depending on the material and the aim of the analysis, specific adaptations are often required because its high chemical reactivity leads to the formation of numerous reactive nitrogen species that make the accurate determination of NO levels difficult. Moreover, the pathogen-induced hypersensitive response leads to high rates of reactive oxygen species production that react with NO and lead to the formation of its oxidized derivates. The aim of this chapter is to provide an overview of the methods that have so far been employed to detect and measure NO in plants during the hypersensitive disease resistance response.

Determining radical penetration of lipid bilayers with new lipophilic spin traps.

Predicting the susceptibility of lipid moieties to radical attack requires a determination of the depth of radical penetration into a lipid membrane. We thus synthesized three homologous series of lipophilic spin traps--DMPO analogs 2-alkanoyl-2-methyl-1-pyrroline N-oxides (11) and PBN derivatives 4-alkoxyphenyl N-tert-butylnitrones (18) and 4-alkoxyphenyl N-admantylnitrones (20). The intercalation depth of these spin traps within the liposomal bilayer was determined via the previously reported NMR technique, which correlates the chemical shift and the micropolarity (measured in ET(30) units) experienced by the pivotal nitronyl carbon. Hydroxyl and alpha-hydroxyalkyl radicals were generated in the extraliposomal aqueous phase and the lowest depth at which a radical could be spin trapped was determined. The ESR data indicate that these radicals can exit the aqueous phase, penetrate the lipid bilayer past the head groups (ET(30)=63 kcal/mol) and the glycerol ester (ET(30)=52 kcal/mol), and pass down to an ET(30) polarity of at least 44 kcal/mol. The latter depth presumably corresponds to the upper portion of the lipid slab. It is likely, if not probable, that having come this far they can abstract the allylic/diallylic hydrogens resident in the midslab at ET(30) values of >31 kcal/mol.

Large atom number Bose-Einstein condensate of sodium.

We describe the setup to create a large Bose-Einstein condensate containing more than 120 x 10(6) atoms. In the experiment a thermal beam is slowed by a Zeeman slower and captured in a dark-spot magneto-optical trap (MOT). A typical dark-spot MOT in our experiments contains 2.0 x 10(10) atoms with a temperature of 320 microK and a density of about 1.0 x 10(11) atoms/cm(3). The sample is spin polarized in a high magnetic field before the atoms are loaded in the magnetic trap. Spin polarizing in a high magnetic field results in an increase in the transfer efficiency by a factor of 2 compared to experiments without spin polarizing. In the magnetic trap the cloud is cooled to degeneracy in 50 s by evaporative cooling. To suppress the three-body losses at the end of the evaporation, the magnetic trap is decompressed in the axial direction.

Immuno-spin trapping analyses of DNA radicals.

Immuno-spin trapping is a highly sensitive method for detecting DNA radicals in biological systems. This technique involves three main steps: (i) in situ and real-time trapping of DNA radicals with the nitrone spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), thus forming DMPO-DNA nitrone adducts (referred to here as nitrone adducts); (ii) purification of nitrone adducts; and (iii) analysis of nitrone adducts by heterogeneous immunoassays using Abs against DMPO. In experiments, DMPO is added prior to the formation of free radicals. It diffuses easily through all cell compartments and is present when DNA free radicals are formed as a result of oxidative damage. Due to its low toxicity, DMPO can be used in cells at high enough concentrations to out-compete the normal reactions of DNA radicals, thus ensuring a high yield of DNA nitrone adducts. Because both protein and DNA nitrone adducts are formed, it is important that the DNA be pure in order to avoid misinterpretations. Depending on the model under study, this protocol can be completed in as few as 6 h.

"Distant spin trapping": a method for expanding the availability of spin trapping measurements.

The technique of spin trapping is used to study a wide range of free radicals in various systems, including those generated in vitro and in vivo. But unfortunately, EPR spectrometers are not always immediately accessible at the site of experimentation, and therefore it is important to find a method that can preserve a radical adduct over longer periods of time. We describe here an alternative method in which the samples can be frozen and transported for EPR measurements at another site. Various spin adducts of DEPMPO were frozen and measured at 0 degrees C at various intervals after freezing to determine their stability in the frozen state. The radical adducts were generated by established methods and stored at two different temperatures; -196 degrees C (liquid nitrogen) and -80 degrees C (dry ice). The experiments were carried out in an aqueous solution with and without a model of reducing environment (2 mM ascorbate). The results indicate that it is feasible to store and transport spin adducts for subsequent analysis. We conclude that this approach, which we term "distant spin trapping", makes it feasible to transport samples to another site for EPR measurements. This should significantly expand the ability to use spin trapping in biology and medicine.

Immuno-spin trapping: detection of protein-centered radicals.

Protein-centered radicals are involved in biological oxidative damage induced by drugs, environmental hazards, and cellular reactive oxygen species. Presently, the technique most widely used to study protein-centered radicals is electron spin resonance (ESR; also known as electron paramagnetic resonance, EPR); used either directly or in combination with the spin-trapping technique. Protein-centered radicals may be trapped with the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) forming DMPO-radical adducts. However, after a few minutes these adducts decay, often by oxidation to DMPO-protein radical-derived nitrone adducts, which are ESR-silent species. Because nitrone adducts are not free radicals and their formation involves the creation of a covalent linkage, they are stable long after the ESR signal decays. In the new alternative technique of immuno-spin trapping, nitrone adducts are detected by using an antibody, i.e., anti-DMPO, that recognizes their nitrone moiety. Immuno-spin trapping is a simple, reliable, affordable, sensitive, and specific approach to detecting protein-centered radicals, and its development brings the power of immunoassays to bear on the field of toxicology of free radical-mediated biological damage.

Evaluation of different combinations of gated trapping, RF-only mode and trap compensation for in-field MALDI Fourier transform mass spectrometry.

MALDI, while providing advantages such as the ability to do in-depth and repeated exploration of the sample, challenges the existing performance capabilities of Fourier transform mass spectrometry (FTMS). The challenge arises because MALDI-produced ions have high mass-to-charge ratios and uncertain kinetic-energy distributions. We demonstrate that a combination of a gated trapping event, a RF-only mode pressure focusing event, and an electrically compensated trap provides a compelling advantage in meeting these challenges. Removal of any of the above combination elements significantly degrades the detection performance of substance P from 850 K resolving power at 34.9 kHz and of melittin from 278 K resolving power at 16.5 kHz when using a 3-Tesla magnet-based spectrometer.

Quantitative spin trapping and triplet quenching by radicals for in vivo studies. I. The chemical model.

In order to determine quantitatively the free radical content and its changes affected by additives using spin trapping under in vivo conditions, an approach is suggested carrying out experiments in a completely mixed open system (CMOS). Measurements have been carried out for a chemical oxidation process as a model system, and analysis of products and of the spin trap was extended by kinetic ESR spectrometry of the spin adducts. Since in a CMOS differential equations of accumulation of all species can be transformed into algebraic expressions using available rate constants for the formation of the spin adducts, corresponding concentrations of free radicals have been calculated. In addition, it has been established that triplet excited photosensitizers have a double effect: increasing the rate of initiation by decomposing hydroperoxide-type compounds and inhibiting the overall process by interactions with free radicals. Results indicate that by changing the "reaction vessel" the method can be applied for ex vivo and in vivo systems.

On the application of 4-hydroxybenzoic acid as a trapping agent to study hydroxyl radical generation during cerebral ischemia and reperfusion.

Aromatic hydroxylation from the reaction between hydroxyl radical and salicylate or its related compounds has been often utilized as a marker for the generation of hydroxyl radicals. We have investigated several technical aspects of applying this method to study hydroxyl radical production during cerebral ischemia and reperfusion using the hydroxylation of 4-hydroxybenzoic acid (4-HBA) to form 3,4-dihydroxybenzoic acid (3,4-DHBA). 4-HBA was administered to rats either through intravenous infusion, or by way of an in vivo microdialysis probe implanted in the brain. Dialysate containing 3,4-DHBA was collected and analyzed by HPLC with electrochemical detection. An endogenous compound was found to co-elute with 3,4 -DHBA but could be separated by varying the chromatographic conditions. Because of interrupted blood flow during cerebral ischemia and reperfusion, delivery of 4-HBA through the microdialysis probe is a preferred method to systemic administration such as intravenous infusion. It is concluded that the oxidation of 4-HBA to 3,4-DHBA can be a reliable and accurate indicator for the formation of hydroxyl radical in vivo if the experiments are well designed to avoid potential pitfalls associated with technical difficulties of the method.

Multisection cerebral blood flow MR imaging with continuous arterial spin labeling.

A method was evaluated to control for off-resonance saturation in noninvasive magnetic resonance imaging with continuous arterial spin labeling of cerebral blood flow. In phantoms and humans, application of amplitude-modulated radio-frequency irradiation during the control image corrected for saturation across the whole brain and made possible cerebral blood flow imaging in multiple sections at arbitrary angles to the labeling plane.

Spin trapping of nitric oxide in aqueous solutions of cigarette smoke.

Nitric oxide, a gaseous free NO radical (.NO) generated in particulate-free gas-phase main-stream smoke of cigarettes, was observed with electrical spin resonance (ESR) using a spin trapping technique. N-Methyl-D-glucamine-dithiocarbamate (MGD)2-Fe2+ complex was used for the NO radical spin trapper in aqueous solution. The intensity of the ESR signal of the spin adduct formed by bubbling smoke from one cigarette increased gradually with time over 2 hours at about 20 degrees C and was constant for 2 days or longer. The time course of the production of the NO radical followed the rate equation y = 1520(1-e-0.018t) for the first-order reaction up to around 25 min after mixing of Fe2+ solution and then slowly approached the maximum value determined by the concentration of the spin adduct. These findings suggest that NO radical is produced slowly from NO radical donors such as amine .NO complexes, peroxinitrite (ONOO-), and other reactants such as nitrogen oxides (NOx), which are produced from the smoke of tobacco leaves, and suggest that its generation could be involved in the decomposition or cleavage of such substances.