alpha-(4-pyridyl-1-oxide)-n-tert-butylnitrone and 1-hydroxyethyl-radical

Hydroxyalkyl radicals have been reported to induce lipid oxidation as the key aspect of the pathogenesis of alcoholic fatty liver disease and are responsible for the alkylation and cleavage of DNA during the metabolism of a wide range of genotoxic compounds. However, relevant kinetic data for the oxidation of unsaturated lipids by 1-hydroxyethyl radical (HER) has not been reported. In this study, the rate constants for the reaction of unsaturated fatty acid methyl esters and sterols with HER have been determined using a competitive kinetic approach employing the spin-trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) as the competitive substrate. Polyunsaturated fatty acid methyl ester is shown to react with HER with an apparent second-order rate constant ranging from (3.7 ± 0.1) × 10(6) L mol(-1) s(-1) for methyl linoleate to (2.7 ± 0.2) × 10(7) L mol(-1) s(-1) for methyl docosahexanoate at 25.0 ± 0.2 °C in ethanol. The apparent second-order rate constant for polyunsaturated fatty acid methyl ester oxidation by HER is dependent on the number of bisallylic hydrogen atoms rather than on the bond dissociation energy value for the weakest C-H bond as determined by ab initio density functional theory calculations. Sterols displayed higher reactivity compared to unsaturated fatty acid methyl esters with apparent second-order rate constants of (2.7 ± 0.1) × 10(6) and (5.2 ± 0.1) × 10(7) L mol(-1) s(-1) at 25.0 ± 0.2 °C in ethanol for cholesterol and ergosterol, respectively. Similar experiments with prenylflavonoids as potential herbal chemopreventive agents for preventing alcoholic liver diseases yield apparent second-order rate constants close to the diffusion control with kapp values of (1.5 ± 0.2) and (3.6 ± 0.1) × 10(9) L mol(-1)s(-1) for 6-prenylnarigerin and xanthohumol at 25.0 ± 0.2 °C in ethanol solution, respectively. Polyunsaturated lipids were revealed to be highly reactive oxidizable substrates toward HER-induced oxidation in biological systems leading to damage of membranes and sensitive structures.

Topics: Ethanol; Fatty Acids, Unsaturated; Flavonoids; Kinetics; Models, Molecular; Pyridines; Sterols; Thermodynamics

The 1-hydroxyethyl radical is a central intermediate in oxidative reactions occurring in beer. The reactivity of thiol-containing compounds toward 1-hydroxyethyl radical was evaluated in beer model solutions using a competitive kinetic approach, employing the spin-trap 4-POBN as a probe and by using electron paramagnetic resonance to detect the generated 1-hydroxyethyl/4-POBN spin adduct. Thiol-containing compounds were highly reactive toward the 1-hydroxyethyl radical with apparent second-order rate constants close to the diffusion limit in water and ranging from 0.5 × 10⁹ L mol⁻¹ s⁻¹ for the His-Cys-Lys-Phe-Trp-Trp peptide to 6.1 × 10⁹ L mol⁻¹ s⁻¹ for the reduced lipid transfer protein 1 (LTP1) isolated from beer. The reactions gave rise to a moderate kinetic isotope effect (k(H)/k(D) = 2.3) suggesting that reduction of the 1-hydroxyethyl radical by thiol-containing compounds takes place by hydrogen atom abstraction from the RSH group rather than electron transfer. The content of reduced thiols in different beers was determined using a previously established method based on ThioGlo-1 as the thiol derivatization reagent and detection of the derivatized thiols by reverse-phase liquid chromatography coupled to a fluorescence detector. The total level of thiol in beer (oxidized and reduced) was determined after a reduction step employing 3,3',3″-phosphanetriyltripropanoic acid (TCEP) as the disulfide reductant. A good correlation among total protein and total thiol content in different beers was observed. The results suggest a similar ratio between reduced thiols and disulfides in all of the tested beers, which indicates a similar redox state.

Topics: Beer; Catalysis; Electron Spin Resonance Spectroscopy; Ethanol; Food Inspection; Food Quality; Food Storage; Free Radical Scavengers; Free Radicals; Kinetics; Models, Chemical; Oxidation-Reduction; Pyridines; Spin Labels; Spin Trapping; Sulfhydryl Compounds

The auto-oxidation of ethanol is likely to proceed via the initial formation of hydroxyethyl radicals (HERs), the one-electron oxidation product. In the laboratory, HERs can be generated by the Fenton reaction (H2O2+ Fe+2) in the presence of ethanol. We report studies on the binding of HERs to serum albumin, generated under Fenton and non-Fenton conditions.. The generation of HER was determined by electron paramagnetic resonance spectroscopy. The formation of ethanol-derived protein adducts was determined by 14C-ethanol incorporation into serum albumin and by the binding of antibodies raised against HER adducts.. We report that serum albumin, used as a model protein, is an effective trapping agent of HERs. In addition, HER radicals covalently bind to albumin to form acid stable adducts. Unexpectedly, we found that under aerobic conditions, the incubation of 50 mM ethanol and phosphate buffer (which contains iron traces) in the absence of the Fenton reagent yields HER radicals as shown by electron paramagnetic resonance spectroscopy and the formation of acid stable protein adducts that are recognized by antibodies raised against HER radical adducts.. Proteins (serum albumin used as a model) are avid trapping agents of HER. There are minimal requirements for the generation of HER, because in the presence of oxygen and a phosphate buffer that contains traces of iron, ethanol readily generates HERs. Thus, HER production is likely to occur in many tissues. The ability of proteins to bind this ethanol radical should be valuable in the diagnosis of alcohol abuse and may be relevant to some of the chronic effects of ethanol.

Topics: Animals; Carbon Radioisotopes; Electron Spin Resonance Spectroscopy; Ethanol; Ferrous Compounds; Free Radicals; Hydrogen Peroxide; Nitrogen Oxides; Oxidation-Reduction; Protein Binding; Pyridines; Rabbits; Serum Albumin; Spin Labels

Impairment of mitochondrial functions has been found in ethanol-induced liver injury. Ethanol can be oxidized to the 1-hydroxyethyl radical (HER) by rat liver microsomal systems. Experiments were carried out to evaluate the ability of HER to cause mitochondrial swelling as an indicator of the mitochondrial permeability transition (MPT). Electron spin resonance (ESR) spectroscopy was used to detect HER and to study its interaction with mitochondria. The ESR signal intensity of the spin adduct formed from alpha-(4-pyridyl-1-oxide) N-tert-butylnitrone (POBN) and HER generated from either a thermic decomposition of 1,1'-dihydroxyazoethane (DHAE) or a Fenton reaction system containing ethanol was markedly diminished by the addition of mitochondria, indicating an interaction between HER and mitochondria. Exposure of rat liver mitochondria to HER generated from either system caused swelling, as reflected by a decrease in absorbance at 540 nm, in a HER concentration-dependent and a cyclosporin A-sensitive manner. Mitochondrial swelling was also induced in the Fenton reaction system without ethanol. The DHAE-dependent generation of HER in mitochondrial suspension resulted in a decrease of membrane protein thiols and collapse of the membrane potential (delta psi). The swelling induced by HER was prevented by glutathione and vitamin E, but not by superoxide dismutase. Catalase did not prevent the swelling caused by the acetaldehyde/hydroxylamine O-sulfonate (HOS) system, but was inhibitory in the Fenton reaction system with or without ethanol. These results indicate that HER, as well as hydroxyl radical, can induce the MPT, and suggest the possibility that the collapse of delta psi caused by HER may, at least in part, contribute to impairment of mitochondrial function caused by ethanol and in ethanol-induced liver injury.

Topics: Animals; Catalase; Cyclosporine; Electron Spin Resonance Spectroscopy; Ethanol; Free Radicals; Intracellular Membranes; Kinetics; Male; Membrane Potentials; Mitochondria, Liver; Mitochondrial Swelling; Nitrogen Oxides; Permeability; Pyridines; Rats; Rats, Wistar; Spectrophotometry; Spin Labels; Superoxide Dismutase; Vitamin E

Extensive ESR spin-trapping studies have shown that ethanol is oxidized to 1-hydroxyethyl radical (HER) by rat and deer mice liver microsomal systems. The ESR detection of POBN/HER nitroxide in bile, and formation of antibodies, which recognize HER adducts in alcoholics, suggest that HER is produced in vivo. In liver, where ethanol is primarily metabolized, only traces of PBN/HER nitroxide are documented. One limitation of the ESR spin-trapping technique, however, is that the nitroxides formed in the presence of cellular reductants can be metabolized to the corresponding ESR "silent" hydroxylamines. Ascorbate and NADPH plus liver microsomes were found to reduce rapidly both POBN/HER and PBN/HER nitroxides to their ESR "silent" hydroxylamine derivatives. An HPLC method with electrochemical detection was developed for the detection and quantification of both POBN/HER and PBN/HER nitroxides, as well as their hydroxylamines. Both the diastereomers of the POBN/HER nitroxide and hydroxylamine can be detected, as can both isomers of the PBN/HER nitroxide, and it is estimated that the sensitivity of the HPLC procedure is in the nM range when using EC detection. The hydroxylamines are stable in ethanol, while pH-dependent auto-oxidation occurs in aqueous buffers. Some of the characteristics associated with HER formation by microsomes as detected with ESR (e.g., sensitivity to SOD and catalase, increase after induction of CYP2E1) are reproduced with the HPLC method. By quantification of the POBN/HER hydroxylamine, the NADPH-dependent rates of HER formation by microsomes from pyrazole-treated rats are estimated to be about 1-1.5 nmol HER per min per mg microsomal protein. This rate is less, as compared to the two electron-dependent rate of acetaldehyde formation by these microsomes, about 10-15 nmol per min per mg protein. Thus, at first approximation, the one electron-dependent rate of ethanol oxidation is about 10% the two electron-dependent rate by isolated microsomes. The HPLC procedure can readily detect the POBN/HER and PBN/HER nitroxides and their hydroxylamine derivatives in the same sample and may be of value in detecting HER spin-trapped adducts under biological reducing conditions.

Topics: Animals; Chromatography, High Pressure Liquid; Cyclic N-Oxides; Cytochrome P-450 CYP2E1; Electron Spin Resonance Spectroscopy; Ethanol; Kinetics; Microsomes, Liver; Nitrogen Oxides; Oxidation-Reduction; Pyridines; Rats; Rats, Sprague-Dawley; Spin Labels

We conducted spin trapping experiments to test the effects of acute and chronic alcohol consumption in livers from rats that had been fed either high fat (35% of energy) or low fat (12% of energy) liquid diets. Rats were anesthetized with isoflurane, the spin trapping agent POBN [alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone] was administered by intravenous injection, and bile samples were collected for electron paramagnetic resonance (EPR) analyses. Two different types of EPR spectra were observed in bile from the animals in these studies. One set of spectral lines was from the 1-hydroxyethyl radical adduct of POBN, which was conclusively identified by injecting the rats with [1-13C]ethanol. The EPR signals of a second type of radical adduct in bile could be observed both before and after acute administration of ethanol. Although the radical(s) responsible for this second series of signals could not be conclusively identified, it is likely that lipid radicals were formed under these conditions and trapped by POBN. For both types of radical adducts, the most intense EPR signals were observed in rats that had been fed alcohol in combination with a high fat diet for 2 wk before the experiments. These results confirm and extend previous data indicating that high levels of dietary fat enhance alcohol-associated free radical formation in the liver.

Topics: Animals; Bile; Dietary Fats; Electron Spin Resonance Spectroscopy; Ethanol; Free Radicals; Liver; Male; Nitrogen Oxides; Pyridines; Rats; Rats, Sprague-Dawley; Spin Labels

Experiments have been designed to reevaluate mechanisms for metabolism of ethanol to 1-hydroxyethyl radicals (HER) in rat liver microsomes. The variables tested include addition of azide, catalase, superoxide dismutase, and deferoxamine, or use of phosphate or Tris buffers. The results indicate that several mechanisms of HER formation are possible, depending on the experimental conditions used to study this process. In the presence of phosphate buffer, which has been used extensively in spite of its ability to chelate iron, HER formation is quite sensitive to changes in hydrogen peroxide availability. These results suggest that Fenton-type reactions produced the oxidizing intermediate responsible for conversion of ethanol to a free radical in phosphate buffer. However, in Tris buffer, HER formation was inhibited markedly by addition of superoxide dismutase, whereas catalase or azide had little effect. These data indicate that the apparent mechanism of radical formation may be influenced by the choice of buffer used. HER formation was almost abolished by the combination of superoxide dismutase and deferoxamine in both buffers, suggesting little enzymatic HER formation by the cytochrome P450 enzymes. When changes in HER formation were compared with rates of ethanol oxidation, it was inferred that 25 to 50% of the acetaldehyde formed during microsomal ethanol oxidation under different experimental conditions could arise via the HER intermediate.

Topics: Animals; Catalase; Deferoxamine; Electron Spin Resonance Spectroscopy; Ethanol; Free Radicals; Kinetics; Male; Microsomes, Liver; Nitrogen Oxides; Pyridines; Rats; Rats, Sprague-Dawley; Sodium Azide; Spin Labels; Superoxide Dismutase

Intravenous administration of the spin-trapping agent alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN) to anesthetized but otherwise untreated rats was used to test for formation of 1-hydroxyethyl radicals in vivo. The only EPR signals observed in bile samples from rats that had received ethanol but no POBN could be attributed to low concentrations of ascorbyl radical. However, when POBN (700 mg/kg, intravenously) was also administered, a nitroxide with a six-line EPR spectrum was readily detected in bile. This spin adduct was proven to be the 1-hydroxyethyl radical adduct of POBN through injection of [1-13C]ethanol to rats, which resulted in the presence of an adduct with a 12-line EPR spectrum. Comparable results were obtained in experiments with isolated perfused rat livers. 1-Hydroxyethyl radical spin adducts of POBN were readily detectable in bile in the presence of only moderate (10-15 mM) concentrations of alcohol. In these experiments, bile samples were collected into a mixture of dipyridyl and bathocuproine disulfonic acid, and the effectiveness of these chelators to prevent ex vivo signal formation was confirmed experimentally. No EPR signals for nitroxide spin adducts were observed in plasma or perfusate, even though high concentrations of POBN and alcohol were present. Taken together, these data indicate that 1-hydroxyethyl radicals are formed in vivo and can be readily detected in bile when high concentrations of POBN are achieved through intravenous injection.

Topics: Animals; Bile; Electron Spin Resonance Spectroscopy; Ethanol; Free Radicals; Injections, Intravenous; Male; Nitrogen Oxides; Pyridines; Rats; Rats, Sprague-Dawley; Spin Labels

Metabolism of ethanol to 1-hydroxyethyl radicals by rat liver microsomes was studied with three nitrone spin trapping agents (POBN, PBN, and DMPO) under essentially comparable conditions. The data indicate that POBN was the superior spin trapping agent for 1-hydroxyethyl radicals, and that DMPO was least efficient. Addition of deferoxamine completely prevented detection of 1-hydroxyethyl radicals with PBN or DMPO, but caused only 50% decrease in EPR signals when POBN was the spin trap. However, superoxide dismutase only decreased 1-hydroxyethyl radical formation when POBN was the spin trap. Other experiments demonstrated that POBN was the most effective of these nitrones for reduction of Fe(III) in aqueous solutions. Furthermore, 1-hydroxyethyl radical adducts were formed when POBN was added to mixtures of ethanol, phosphate buffer, POBN and FeCl3, but this effect did not occur with either PBN or DMPO. Thus, these data indicate that undesirable effects of POBN on iron chemistry may influence results of spin trapping experiments, and complicate interpretation of the resulting data.

Topics: Animals; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Ethanol; Free Radicals; Male; Microsomes, Liver; Nitrogen Oxides; Pyridines; Rats; Rats, Sprague-Dawley; Spin Labels; Superoxide Dismutase