Colorimetric alcohol assays with alcohol oxidase

A method is described for manufacturing crude alcohol oxidase (EC 1.1.3.13) preparations which are suitable for application in colorimetric alcohol assays. The procedure involves a one-step removal of catalase activity from a partially purified preparation of alcohol oxidase from the yeast Hansenula polymorpha via dialysis against 3-amino-1,2,4-triazole and hydrogen peroxide. Thus, the irreversible inactivation of more than 90% of the catalase present was achieved, which is prerequisite for the use of alcohol oxidase preparations in colorimetric alcohol assays via peroxidase-mediated oxidation of dyes. This type of assay was shown to be rapid, accurate and sensitive. The influence of the relative concentrations of the various assay constituents such as alcohol oxidase, catalase and peroxidase is discussed. It is concluded that this colorimetric alcohol assay is particularly suitable for the determination of ethanol in fermentation broths, both in qualitative and in quantitative tests.     

Coniferyl alcohol metabolism in conifers -- II. Coniferyl alcohol and dihydroconiferyl alcohol biosynthesis.

Coniferaldehyde and NADPH when incubated with microsomes isolated from developing xylem of Pinus strobus yielded coniferyl alcohol and dihydroconiferyl alcohol in vitro. D-(+)-Pinitol was also found to be a microsomal constituent. Endogenous E-coniferyl alcohol content, quantified in dormant buds, cambium, bark and needles of Pinus resinosa and P. strobus by isotope-dilution combined gas chromatography mass spectrometry (GC/MS) using ring-(13)C(6)-coniferyl alcohol, was at a level similar to that of endogenous indol 3-ylacetic acid (IAA). Wounding (branch girdling) induced more than a 10-fold increase in content of endogenous E-coniferyl alcohol in dormant non-lignifying cambium, a clear indication that monolignol biosynthesis is not coupled to lignification.     

Synergism between coenzyme and alcohol binding to liver alcohol dehydrogenase

Heterotropic cooperativity effects in the binding of alcohols and NAD+ or NADH to liver alcohol dehydrogenase have been examined by equilibrium measurements and stopped-flow kinetic studies. Equilibrium data are reported for benzyl alcohol, 2-chloroethanol, 2,2-dichloroethanol, and trifluoroethanol binding to free enzyme over the pH range 6-10. Binary-complex formation between enzyme and alcohols leads to inner-sphere coordination of the alcohol to catalytic zinc and shows a pH dependence reflecting the ionization states of zinc-bound water and the zinc-bound alcohol. The affinity of the binding protonation state of the enzyme for unionized alcohols increases approximately by a factor of 10 on complex formation between enzyme and NAD+ or NADH. The rate and kinetic cooperativity with coenzyme binding of the alcohol association step indicates that enzyme-bound alcohols participate in hydrogen bonding interactions which affect the rates of alcohol and coenzyme equilibration with the enzyme without providing any pronounced contribution to the net energetics of alcohol binding. The pKa values determined for alcohol deprotonation at the binary-complex level are linearly dependent on those of the free alcohols, and can be readily reconciled with the pKa values attributed to ionization of zinc-bound water. Alcohol coordination to catalytic zinc provides a major contribution to the pKa shift which ensures that the substrate is bound predominantly as an alcoholate ion in the catalytically productive ternary complex at physiological pH. The additional pKa shift contributed by NAD+ binding is less pronounced, but may be of particular mechanistic interest since it increases the acidity of zinc-bound alcohols relatively to that of zinc-bound water.     

Kinetic characteristics of n-butyl alcohol and iso-butyl alcohol in a composite bead air biofilter

Kinetic characteristics of n-butyl alcohol and iso-butyl alcohol in a composite bead biofilter were investigated. The microbial growth rate of n-butyl alcohol was greater than that of iso-butyl alcohol in the average inlet concentration range of 50-300 ppm. The microbial growth rate was inhibited at higher inlet concentration, and the inhibitive effect in the concentration range of 50-150 ppm was more pronounced than that in the concentration range of 150-300 ppm. The degree of inhibitive effect for n-butyl alcohol was more sensitive than that for iso-butyl alcohol in the concentration range of 50-150 ppm. The zero-order kinetic with the diffusion rate limitation could be regarded as the most adequate biochemical reaction model. The biodegradation rate of n-butyl alcohol was greater than that of iso-butyl alcohol in the average inlet concentration range of 50-300 ppm. The biochemical reaction rate was also inhibited at higher inlet concentration, and the inhibitive effect for iso-butyl alcohol was more pronounced than that for n-butyl alcohol. The factor of the chemical structure of compound was more predominant in the microbial growth and biochemical reaction processes. The maximum elimination capacity of n-butyl alcohol and iso-butyl alcohol were 55.7 and 34.8 g C h(-1)m(-3) bed volume, respectively. The compound with no side group in the main chain would be easier biodegraded by the microbial.     

Quinone-dependent alcohol dehydrogenases and fad-dependent alcohol oxidases

This review considers quinone-dependent alcohol dehydrogenases and FAD-dependent alcohol oxidases, enzymes that are present in numerous methylotrophic eu- and prokaryotes and significantly differ in their primary and quaternary structure. The cofactors of the enzymes are bound to the protein polypeptide chain through ionic and hydrophobic interactions. Microorganisms containing these enzymes are described. Methods for purification of the enzymes, their physicochemical properties, and spatial structures are considered. The supposed mechanism of action and practical application of these enzymes as well as their producers are discussed.     

Effects of alcohol mixed with energy drink and alcohol alone on subjective intoxication

Recent studies suggest that the combination of caffeine-containing drinks together with alcohol might reduce the subjective feelings of alcohol intoxication—the so-called “masking effect”. In this study, we aimed to review the effects of alcohol in combination with caffeine or energy drink with special focus on the “masking effect”. Fifty-two healthy male volunteers were analysed concerning breath alcohol concentration and subjective sensations of intoxication using a 18 item Visual Analogue Scale in a randomised, double-blinded, controlled, four treatments cross-over trial after consumption of (A) placebo, (B) alcohol (vodka 37.5 % at a dose of 46.5 g ethanol), (C) alcohol in combination with caffeine at a dose of 80 mg (equivalent to one 250 ml can of energy drink) and (D) alcohol in combination with energy drink at a dose of 250 ml (one can). Primary variables were headache, weakness, salivation and motor coordination. Out of four primary variables, weakness and motor coordination showed a statistically significant difference between alcohol and non-alcohol group, out of 14 secondary variables, five more variables (dizziness, alterations in sight, alterations in walking, agitation and alterations in speech) also showed significant differences due mainly to contrasts with the non-alcohol group. In none of these end points, could a statistically significant effect be found for the additional ingestion of energy drink or caffeine on the subjective feelings of alcohol intoxication. This within-subjects study does not confirm the presence of a “masking effect” when combining caffeine or energy drink with alcohol.     

Biodegradation kinetic behaviors of n-butyl alcohol and sec-butyl alcohol in a composite bead biofilter

Biodegradation kinetic behaviors of n-butyl alcohol and sec-butyl alcohol in a composite bead biofilter were investigated. The microbial growth rate of n-butyl alcohol was greater than that of sec-butyl alcohol in the inlet concentration range of 50–300 ppm. The microbial growth rate was inhibited at higher inlet concentration, and the inhibitive effect in the concentration range of 50–150 ppm was more pronounced than that in the concentration range of 150–300 ppm. The degree of inhibitive effect for n-butyl alcohol was more sensitive than that for sec-butyl alcohol in the concentration range of 50–150 ppm. The zero-order kinetic with the diffusion rate limitation could be regarded as the most adequate biochemical reaction model. For the biochemical reaction process, the biochemical reaction rate coefficient of n-butyl alcohol was greater than that of sec-butyl alcohol in the inlet concentration range of 50–300 ppm. The biochemical reaction rate coefficient was decreased with increasing inlet concentration. The inhibitive effect for sec-butyl alcohol was more pronounced than that for n-butyl alcohol. The factor of the chemical structure of compound was more predominant in the microbial growth and biochemical reaction processes. The maximum elimination capacity of n-butyl alcohol and sec-butyl alcohol were 55.7 and 20.9 g C h^?1 m^?3 bed volume, respectively. The primary alcohol was easily biodegraded by the microbial.     

Clinical versus laboratory detection of alcohol abuse: the alcohol clinical index.

To determine reliable indicators of alcohol abuse a comprehensive set of clinical and laboratory information was acquired from three groups of subjects with a wide range of drinking histories: 131 outpatients with alcohol problems, 131 social drinkers, and 52 patients from family practice. Findings from clinical examination provided greater diagnostic accuracy than laboratory tests for detecting alcohol abuse. Logistic regression analysis produced an overall accuracy of 85-91% for clinical signs, 84-88% for items from the medical history, and 71-83% for laboratory tests in differentiating the three groups. Further analyses showed 17 clinical signs and 13 medical history items that formed a highly diagnostic instrument (alcohol clinical index) that could be used in clinical practice. A probability of alcohol abuse exceeding 0.90 was found if four or more clinical signs or four or more medical history items from the index were present. Despite recent emphasis on the laboratory diagnosis of alcohol abuse simple clinical measures seem to provide better diagnostic accuracy.     

Stereospecificity of cinnamyl alcohol dehydrogenase and synthesis of stereospecifically labelled coniferyl alcohol

Using horse liver alcohol dehydrogenase, stereospecifically tritiated (R)- and (S)-(γ-³H)-coniferyl alcohol was synthesized. Using both of these substrates it was demonstrated that cinnamyl alcohol dehydrogenase from lignifying Forsythia tissue specifically removes the pro-R-hydrogen atom of coniferyl alcohol in the oxidation to the aldehyde. This also means that in the reverse reaction the A-hydrogen of NADPH is transferred to the Re-site of coniferyl aldehyde.     

Liver alcohol dehydrogenase

The article deals with the structure and function of liver alcohol dehydrogenase and reviews mainly literature published after 1979, i.e., summarizes progress made in the field since Klinman presented her review on alcohol dehydrogenases. The emphasis will be on high-resolution crystallographic data, results obtained with metal-substituted enzyme derivatives, and on the mechanism and pH dependence of the catalytic reaction.     

Evaluation of un-medicated, self-paced alcohol withdrawal

It is currently unclear how effective un-medicated, self-paced alcohol withdrawal is in reducing alcohol consumption in alcohol dependent clients. To address this question, the current study examined the reduction in alcohol consumption, assessed by breath alcohol and drink diary self-report, of 405 alcohol-dependent clients over a 10-day, un-medicated, self-paced alcohol reduction program that included group discussion of strategies for titrating between withdrawal and intoxication. It was found that attendance at treatment sessions was associated with a reduction in alcohol consumption, reflected in both breath alcohol and diary measures, and these two measures were significantly correlated. Overall, 35% of clients achieved a zero breath alcohol reading by their final session, although this percentage increased to 56% of clients who attended all 10 sessions. Withdrawal seizures occurred in only 0.5% of clients despite 17.2% having a history of seizures in other settings. It is concluded that the alcohol reduction protocol outlined here provides an effective and safe method for reducing alcohol consumption in severely alcohol dependent clients, and that methods for augmenting attendance, such as contingency management, should enhance the effectiveness of this treatment.     

Novel substrates of yeast alcohol dehydrogenase--4. Allyl alcohol and ethylene glycol

In the present work, we have determined the steady-state kinetic constants for yeast alcohol dehydrogenase-catalyzed oxidation of allyl alcohol (H2C = CH.CH2OH) and ethylene glycol (HOCH2.CH2OH) with NAD+, at pH 8.0; also, a kinetic mechanism for the former reaction was determined at the same pH. In addition, it was found that acrolein is a potent inhibitor of yeast alcohol dehydrogenase.     

Thiochrome inhibition of alcohol dehydrogenase]

It is shown that thiochrome inhibits alcohol dehydrogenase. Thiochrome is able to be bound with alcohol dehydrogenase more quickly than other thiamine metabolites. This process is specific and has common features with the process of NAD binding by this enzyme. The inhibition of alcohol dehydrogenase by thiochrome is concurrent to NAD. The constant of alcohol dehydrogenase inhibition by thiochrome is 3.9 x 10(-5) M.     

The heart and alcohol

The cardiovascular effects of the ingestion of ethyl alcohol are determined by the amount consumed and time factors as well as the nutritional status of the individual. Acute alcoholism produces various cardiac manifestations that are related primarily to the concentration of alcohol in the blood. Chronic alcoholism is associated with three identifiable cardiovascular syndromes that have been designated alcoholic myocardosis, nutritional heart disease and beriberi heart disease. Differentiation is indicated because of their respective distinguishing diagnostic features and prognostic implications. The therapeutic effects of alcohol in coronary artery disease are apparently attributable to cerebral responses rather than demonstrable increase in coronary blood flow.     

Genetics and genomics of alcohol sensitivity

Alcohol abuse and alcoholism incur a heavy socioeconomic cost in many countries. Both genetic and environmental factors contribute to variation in the inebriating effects of alcohol and alcohol addiction among individuals within and across populations. From a genetics perspective, alcohol sensitivity is a quantitative trait determined by the cumulative effects of multiple segregating genes and their interactions with the environment. This review summarizes insights from model organisms as well as human populations that represent our current understanding of the genetic and genomic underpinnings that govern alcohol metabolism and the sedative and addictive effects of alcohol on the nervous system.     

Production of a perillyl alcohol dehydrogenase by site-directed mutagenesis of a benzyl alcohol dehydrogenase

Benzyl alcohol dehydrogenase from Acinetobacter calcoaceticus oxidises a wide range of aromatic and other cyclic alcohols and it has high specificity constants for these substrates, but it does not oxidise short- or long-chain aliphatic alcohols. Mutation of an active-site arginine to a histidine can switch the substrate specificity of the enzyme so that it has a very much greater preference for perillyl alcohol than for benzyl alcohol. © Rapid Science Ltd. 1998     

Further investigations of the transient kinetics of alcohol oxidation catalysed by horse liver alcohol dehydrogenase

Due to the controversy over the half-of-the-sites reactivity of horse liver alcohol dehydrogenase during benzyl alcohol oxidation, we have re-investigated the transient kinetics, stoichiometry and rate parameters over a wide range of substrate concentrations (0.05 mm to 40 mm) at pH 7.0 and 8.5 and using newly determined extinction coefficients. Data were elaborated by computer analysis in order to separate the initial rapid step (burst) from the whole time-course of the reaction. It has been found that: (1) the dependence of the burst amplitude upon benzyl alcohol concentration is distinctly biphasic. In the range from 0.05 mm up to approximately 1 mm the burst amplitude is rather insensitive to changes in alcohol concentration and corresponds to 50% of the active sites of the enzyme; for alcohol concentrations greater than 1 mm this amplitude increases and reaches a value of approximately 90% when benzyl alcohol is 40 mm. (2) The steady-state initial rate is also biphasic with respect to alcohol concentration, indicative of substrate inhibition, which begins in the concentration range at which deviation from the half-burst also appears. In other words, burst amplitudes larger than 50% are concomitant with inhibition of the rate of enzyme turnover. (3) In the presence of isobutyramide the burst is larger than 50% for the whole range of concentration of the substrate and extrapolates at infinite substrate concentration to approximately 90% of the enzyme sites. (4) With deuteroethanol as substrate, the burst is larger than 50%, with or without isobutyramide, and extrapolates to approximately 95% of the enzyme sites at infinite substrate concentration. These data explain the discrepancy of results in the literature concerning the transient kinetics of alcohol oxidation. Mechanistic implications of the results (particularly the deviation from the halfof-the-sites behaviour of benzyl alcohol under inhibition conditions) are discussed.     

Aliphatic alcohol dehydrogenase from potato tubers

Potato tubers are shown to contain at least 3 alcohol dehydrogenases, one active with NAD and aliphatic alcohols, one active with NADP and terpene alcohols and one active with NADP and aromatic alcohols. The purification of the aliphatic alcohol dehydrogenase is described and its activity with a wide range of substrates is reported. On the basis of substrate specificity, the enzyme is shown to resemble yeast alcohol dehydrogenase rather than liver alcohol dehydrogenase. The enzyme shows high activity with and high affinity for ethanol, activity and affinity decline as the chain length is increased from ethanol to butanol, but a further increase in chain length leads to increased affinity for the alcohol. The physiological significance of the results is briefly discussed.     

Effect of alcohol on hepatocyte mitochondria

The authors studied ultrastructural changes of hepatocytes under the effect of alcohol. The greatest changes were revealed in the mitochondria. Physical activity and hypoprotein diet produced a significant influence on the results of alcohol poisoning. The first factor reduced, and the second enhanced the deleterious effect of alcohol.     

Characteristics of alcohol/polyol dehydrogenases. The zinc-containing long-chain alcohol dehydrogenases

Sixteen characterized alcohol dehydrogenases and one sorbitol dehydrogenase have been aligned. The proteins represent two formally different enzyme activities (EC 1.1.1.1 and EC 1.1.1.14), three different types of molecule (dimeric alcohol dehydrogenase, tetrameric alcohol dehydrogenase, tetrameric sorbitol dehydrogenase), metalloproteins with different zinc contents (1 or 2 atoms per subunit), and polypeptide chains from different kingdoms and orders (mammals, higher plants, fungus, yeasts). Present comparisons utilizing all 17 forms reveal extensive variations in alcohol dehydrogenase, but with evolutionary changes that are of the same order in different branches and at different times. They emphasize the general importance of particular residues, suggesting related overall functional constraints in the molecules. The comparisons also define a few coincidences between intron positions in the genes and gap positions in the gene products. Only 22 residues are strictly conserved; half of these are Gly, and most of the remaining ones are Pro or acidic residues. No basic residue, no straight-chain hydrophobic residues, no aromatic residues, and essentially no branched-chain or polar neutral residues are invariable. Tentative consensus sequences were calculated, defining 13 additional residues likely to be typical of but not invariant among the alcohol dehydrogenases. These show a predominance of Val, charged residues, and Gly. Combined, the comparisons, which are particularly relevant to the data base for protein engineering, illustrate the requirements for functionally important binding interactions, and the extent of space restrictions in proteins with related overall conformations and functions.