Mechanism Of Ethanol Oxidation Research Articles

A novel method for evaluation of ethanol oxidation in living brain

We propose a method for intravital examination of ethanol metabolism in rat brain. Calipsol-anesthetized rats were fixed in stereotaxis and ethanol solution was infused into the lateral cerebral ventricle via an orifice in the skull at a constant rate with a syringe or micro-pump. Elimination of ethanol and production of acetaldehyde (ethanol metabolite) were measured in perfusate samples from the cisterna magna by gas chromatography. The method is highly sensitive, reliable, and reproducible and allows to study the kinetics and enzyme mechanisms of ethanol oxidation in the brain and regulation of this process.

Enzymatic Mechanisms of Ethanol Oxidation in the Brain

The exact enzymatic mechanisms of ethanol oxidation in the brain are still unclear. The catalase-mediated oxidation of ethanol was demonstrated in rat brain using incubation of brain homogenates with catalase inhibitors. The role of the alcohol dehydrogenase (ADH) or cytochrome P450-dependent system in this process is possible, but has not been confirmed. The objective of the study was to determine the contribution of the different enzymatic pathways to ethanol oxidation in brain homogenates from mice and rats. Three approaches were used to investigate the enzymatic mechanisms of ethanol oxidation in the brain of rats and mice: (1) preincubation of brain homogenates with inhibitors of the ethanol-metabolizing enzymes (catalase, CYP2E1, ADH, and ALDH); (2) utilization of mice with genetic deficiency in ethanol-metabolizing enzymes (catalase, CYP2E1, or both enzymes); and (3) determination of ethanol oxidation in brain subcellular fractions known to have differential activity of ethanol-metabolizing enzymes. The ethanol-derived acetaldehyde (AC) and acetate were determined in brain samples by gas chromatography. The catalase inhibitors sodium azide (5 mM) and aminotriazole (5 mM) as well as CYP2E1 inhibitors diallyl sulfide (2 mM) and beta-phenethyl isothiocyanate (0.1 mM) lowered significantly the accumulation of the ethanol-derived AC and acetate in brain homogenates. The ADH inhibitor 4-methyl pyrazole (5 mM) significantly decreased the acetate but not the AC accumulation. Ethanol-derived AC accumulation in brain homogenates of acatalasemic mice was 47% of the control value, 91% in CYP2E1-null mice, and 24% in double mutants (with deficiency of both catalase and CYP2E1). The highest levels of ethanol oxidation were found in microsomal and peroxisomal subcellular brain fractions, where CYP2E1 and catalase are located, respectively. Catalase is the key enzyme of ethanol oxidation in the brain of rodents: it may be responsible for about 60% of the process. CYP2E1 plays an important role in ethanol oxidation in the rodent brains. Alcohol dehydrogenase plays a minor role, if any, in this process. Aldehyde dehydrogenase plays the crucial role in the further oxidation of ethanol-derived AC in the brain homogenates.

Effect of temperature on the mechanism of ethanol oxidation on carbon supported Pt, PtRu and Pt 3Sn electrocatalysts

The electrochemical oxidation of ethanol on carbon supported Pt, PtRu and Pt 3Sn catalysts was studied in acid solutions at room temperature and in direct ethanol fuel cells (DEFC) in the temperature range 70–100 °C. In all the experiments, an enhancement of the activity for the ethanol oxidation was observed on the binary catalysts. In acid solution the improvement at low current densities was higher on PtRu than on Pt 3Sn. In DEFC tests, at 70 °C the cells with PtRu and Pt 3Sn showed about the same performance, while for T > 70 °C the cells with Pt 3Sn as anode material performed better than those with PtRu as anode material. The apparent activation energy for ethanol oxidation on PtRu catalyst was lower than on Pt 3Sn, particularly at high cell potentials, i.e. at low current densities. At low temperatures and/or low current densities, the positive effect of Ru oxides on the bifunctional mechanism determined the enhancement of activity for the ethanol oxidation reaction, while at high temperatures the positive effect of Sn alloying (enlarged lattice parameter) on CH 3CH 2OH adsorption and C–C cleavage prevails.

The influence of nitric oxide on the oxidation of methanol and ethanol

Ethanol has become a promising biofuel, widely used as a renewable fuel and gasoline additive. Describing the oxidation kinetics of ethanol with high accuracy is required for the development of future efficient combustion devices with lower pollutant emissions. The oxidation process of ethanol, from reducing to oxidizing conditions, and its pressure dependence (20, 40 and 60 bar) has been analyzed in the 500–1100 K temperature range, in a tubular flow reactor under well controlled conditions. The effect of the presence of NO has been also investigated. The experimental results have been interpreted in terms of a detailed chemical kinetic mechanism with the GADM mechanism (Glarborg P, Alzueta MU, Dam-Johansen K and Miller JA, 1998) as a base mechanism but updated, validated, extended by our research group with reactions added from the ethanol oxidation mechanism of Alzueta and Hernández (Alzueta MU and Hernández JM, 2002), and revised according to the present high-pressure conditions and the presence of NO. The final mechanism is able to reproduce the experimental trends observed on the reactants consumption and main products formation during the ethanol oxidation under the conditions studied in this work. The results show that the oxygen availability in the reactant mixture has an almost imperceptible effect on the temperature for the onset of ethanol consumption at a constant pressure, but this consumption is faster for the highest value of air excess ratio (λ) analyzed. Moreover, as the pressure becomes higher, the oxidation of ethanol starts at lower temperatures. The presence of NO promotes ethanol oxidation, due to the increased relevance of the interactions of CH3 radicals and NO2 (from the conversion of NO to NO2 at high pressures and in presence of O2) and the increased concentration of OH radicals from the interaction of NO2 and water.

Ethanol Electro‐oxidation in Ruthenium‐Oxide‐Coated Titanium Electrodes

A systematic investigation of ethanol oxidation on electrodes has been carried out in acid solution. The experimental results show high selectivity toward acetaldehyde formation. One of the secondary oxidation products of ethanol forms a blocking film at the electrode surface which can be removed during electrosynthesis using pulse control of the working potential. The influence of the electrolysis conditions, the electrode mechanism of ethanol oxidation, and RΩ values measured under pseudo‐Tafel plots are discussed.

Particle Size and Surface Chemistry in Photoelectrochemical Reactions at Semiconductor Particles

In the present paper reactions at small and large ZnS particles have been investigated. It has been shown that ethanol is selectively oxidized at large (micrometer) particles to acetaldehyde without side products by a “two hole” process. In the case of nanometer particles the primarily formed α-hydroxyethyl radicals in a “one hole” process undergo a secondary reaction, i.e., the dimerization and disproportionation of the free radicals. It has been shown that a two hole process on nanometer particles becomes impossible because the time interval between two successive photon absorption incidents which lead to a successful hole transfer process in a 1-nm particle is much longer than the maximum lifetime of the α-hydroxyethyl radicals formed in the first step. The different mechanisms of ethanol oxidation and the influence of surface chemistry are discussed in detail.

Kinetics for Electro‐oxidation of Ethanol on Thermally Prepared Ruthenium Oxide in Alkaline Solution

The electro‐oxidation of ethanol on thermally prepared ruthenium oxide in alkaline solution was investigated by rotating‐disk electrode (RDE) and polarization techniques. The oxidation of ethanol was mediated by perruthenate electrogenerated on the electrode surface. The following mechanism of ethanol oxidation on ruthenium oxide was proposed Heterogeneous reactionsHomogeneous reactionThe kinetic parameters also were obtained by comparing the developed kinetic model and the experimental results.

Kinetics of Ethanol Oxidation on Electroless Ni ‐ P / SnO2 / Ti Electrodes in KOH Solutions

The electrolytic oxidation of ethanol was investigated on the electroless electrodes in . The mechanisms of ethanol oxidation on the electrode were studied via cyclic voltammetry and polarization curves. The kinetic equations were derived, and the kinetic parameters were obtained from a comparison of experimental results and the kinetic equations. The redox reaction of the nickel electrode in the alkaline solution is reversible, and the rate constants related to this reaction, as well as , are functions of applied potential. Ethanol oxidation on the electroless is through a chemical reaction with a rate constant . These rate constants were determined from the current‐potential curves and the kinetic model at various concentrations of ethanol. The rate constants for electrochemical reaction could be expressed as , , where was the applied potential vs. , and the chemical reaction rate constant was.

Molecular orbital studies of enzyme mechanisms. II. Catalytic oxidation of alcohols by liver alcohol dehydrogenase

AbstractSemiempirical (AM1) molecular orbital theory has been used to investigate the oxidation of alcohols at the active site of liver alcohol dehydrogenase (LADH). The model active site consists of a zinc dication coordinated to two methyl‐mercaptans (Cys‐46, Cys‐176), an imidazole (His‐67), and a water. An imidazole (His‐51) hydrogen bonded to a hydroxy‐acetate (Ser‐48) forms the remote base. AM1 calculations that address the two distinct steps in the catalytic mechanism of ethanol oxidation by LADH are reported. These two steps are: (1) the deprotonation of ethanol by imidazole (His‐51) via hydrogen‐bonded hydroxy‐acetate (Ser‐48), creating a proton relay system; and (2) the rate‐limiting hydride transfer step from ethanol C1 to nicotinamide adenine dinucleotide (NAD+), leading to product formation. Detailed calculations have been used to resolve the unsolved problems of mechanisms that have been suggested on the basis of kinetic data and crystal structures of several LADH complexes. We investigated two possible mechanisms for the deprotonation of ethanol, by zinc‐bound OH− and by direct deprotonation of zinc‐bound ethanol by imidazole via hydroxyacetate (Ser‐48). Our calculations show that there is no need for LADH to activate a water molecule at the active site as in many other zinc enzymes. This result agrees with experimental evidence. Our calculations also indicate that substrates are bound in an inner‐sphere‐pentacoordinated complex to the active site zincion. In this case, spectroscopic investigations agree with our results but crystallographic data do not. The highest activation energy is found for the hydride transfer, in agreement with the experiment. Finally, we proposed an alternative mechanism for the mode of action of LADH based upon our results. © 1993 John Wiley & Sons, Inc.

Temperature dependence of the microsomal oxidation of ethanol by cytochrome P450 and hydroxyl radical-dependent reactions

The temperature dependence and activation energies for the oxidation of ethanol by microsomes from controls and from rats treated with pyrazole was evaluated to determine whether the overall mechanism for ethanol oxidation by microsomes was altered by the pyrazole treatment. Arrhenius plots of the temperature dependence of ethanol oxidation by pyrazole microsomes were linear and exhibited no transition breaks, whereas a slight break was observed at about 20 ± 2.5 °C with control microsomes. Energies of activation (about 15–17 kcal/mol) were identical for the two microsomal preparations. Although transition breaks were noted for the oxidation of substrates such as dimethylnitrosamine and benzphetamine, activation energies for these two substrates were similar for control microsomes and microsomes from the pyrazole-treated rats. The addition of ferric-EDTA to the microsomes increased the rate of ethanol oxidation by a hydroxyl radical (.OH)-dependent pathway. Arrhenius plots of the .OH-dependent oxidation of ethanol by both microsomal preparations were linear with energies of activation (about 7 kcal/mol) that were considerably lower than values found for the P450-dependent pathway. These results suggest that, at least in terms of activation energy, the increase in microsomal ethanol oxidation by pyrazole treatment is not associated with any apparent change in the overall mechanism or rate-limiting step for ethanol oxidation but likely reflects induction of a P450 isozyme with increased activity toward ethanol. The lower activation energy for the .OH-dependent oxidation of ethanol suggests that different steps are rate limiting for oxidation of ethanol by .OH and by P450, which may reflect the different enzyme components of the microsomal electron transfer system involved in these reactions.

Oxygen radical generation by microsomes: Role of iron and implications for alcohol metabolism and toxicity

Experiments were carried out to evaluate whether the molecular mechanism for ethanol oxidation by mcirosomes, a major pathway of alcohol metabolism, involved generation of hydroxyl radical (·OH). Microsomes oxidized chemical ·OH scavengers (KMB, DMSO, t-butyl alchol, benzoate) by a reaction sensitive to catalase, but not SOD. Iron was required for microsomal ·OH generation in view of the potent inhibition by desferrioxamine; however, the chelated form of iron was important. Microsomal ·OH production was effectively stimulated by ferric EDTA or ferric DTPA, but poorly increased with ferric ATP, ferric citrate, or ferric ammonium sulfate. By contrast, the latter ferric complexes effectively increased microsomal chemiluminescence and lipid peroxidation, whereas ferric EDTA and ferric DTPA were inhibitory. Under conditions that minimize ·OH production (absence of EDTA, iron) ethanol was oxidized by a cytochrome P-450-dependent process independent of reactive oxygen intermediates. Under conditions that promote microsomal ·OH production, the oxidation of ethanol by ·OH becomes more significant in contributing to the overall oxidation of ethanol by microsomes. Experiments with inhibitors and reconstituted systems containing P-450 reductase indicated that the reductase is the critical enzymes locus for interacting with iron and catalyzing production of reactive oxygen species. Mirosomes isolated from rats chronically fed ethanol catalyzed oxidation ·OH scavengers, light emission and inactivation of added metabolic enzymes at elevated rates, and displayed an increase in ethanol oxidation by a ·OH-dependent and a P-450-dependent pathway. It is possible that enhanced generation of reactive oxygen intermediates by microsomes may contribute to teh hepatotoxic effects of ethanol.

Kinetics and thermodynamics of ethanol oxidation catalyzed by genetic variants of the alcohol dehydrogenase from Drosophila melanogaster and D. simulans

Four naturally occurring variants of the alcohol dehydrogenase enzyme (ADH; EC 1.1.1.1) from Drosophila melanogaster and D. simulans, with different primary structures, have been subjected to kinetic studies of ethanol oxidation at five temperatures. Two amino acid replacements in the N-terminal region which distinguish the ADH of D. simulans from the three ADH allozymes of D. melanogaster generate a significantly different activation enthalpy and entropy, and Gibbs free energy change. The one or two amino acid replacements in the C-terminal region between the ADH allozymes of D. melanogaster do not have such clear-cut effects. All four ADH variants show highly negative activation entropies. Sarcosine oxidation by the ADH-71k variant of D. melanogaster has an activation energy barrier similar to that of ethanol oxidation. Three amino acid differences between the ADH of D. simulans and the ADH-F variant of D. melanogaster influence the k cat and k cat/ K m eth constant by a maximum factor of about 2 and 2.5, respectively, over the whole temperature range. Product inhibition patterns suggest a ‘rapid equilibrium random’ mechanism of ethanol oxidation by the ADH-71k, and the ADH of D. simulans.

Formaldehyde and Acetaldehyde in Exhaust Gases Emitted from an Ethanol Fueled S. I. Engine : Clarification of the Formation Mechanism by Reactor Experiment

The formation conditions of intermediate products in exhaust gases from an ethanol fueled S. I. engine were investigated. The experiments were performed at temperatures of 650-900 K, residence times 0-100 ms, and an air fuel equivalence ratio range of 0.8-1.4. The concentrations of acetaldehyde, formaldehyde, or hydrocarbons produced by oxidation of unburned ethanol, change with the residence time and the gas range at residence times of 40-60 ms. The conversion of nitric oxide to nitrogen dioxide played a significant role in the formation and consumption of aldehyde as in the case of methanol oxidation. A suggested aldehyde formation path was obtained by a produced a reaction mechanism of ethanol oxidation, which mainly consisted of hydrogen atom abstraction reactions.

Kinetic model of ethanol oxidation on supported CuO catalyst

A probable mechanism of ethanol oxidation to aœtaldehyde on supported CuO catalyst is suggested together with the kinetic equations based on experimental data.

The effect of dimethylsulfoxide and other hydroxyl radical scavengers on the oxidation of ethanol by rat liver microsomes

The NADPH-dependent oxidation of ethanol by rat liver microsome preparations was studied in the presence of azide to inhibit the peroxidatic activity of catalase. Dimethylsulfoxide, benzoate, mannitol and thiourea, four compounds that react rapidly with hydroxyl radicals, each inhibited the oxidation rate of ethanol. Inhibition was competitive with respect to ethanol. In contrast, urea, a compound that reacts poorly with hydroxyl radicals, was essentially without effect. Dimethylsulfoxide at concentrations that inhibited the oxidation of ethanol had no effect on the xanthine oxidase-mediated oxidation of ethanol or on aniline hydroxylase or aminopyrine demethylase activity of microsomes. These results suggest that ethanol oxidation by microsomes can be dissociated from drug metabolism and that the mechanism of ethanol oxidation may involve, in part, the interaction of ethanol with hydroxyl radicals that are generated by microsomes during the oxidation of NADPH.

The stability of ethanol in stored blood: Part II. The mechanism of ethanol oxidation

The oxidation of ethanol in stored human blood has been investigated. The oxidizing activity is shown to arise from an oxyhaemoglobin intermediate and to be inhibited by compounds which destroy oxyhaemoglobin. The reaction is first order with respect to oxyhaemoglobin concentration and zero order with respect to ethanol concentration. The activation energy for the production of the intermediate is calculated from an Arrhenius plot as 25 kcal mole -1. An overall mechanism is proposed for blood ethanol oxidation and the stabilization of blood ethanol solutions is discussed.