Alcohol and acetaldehyde in rat's milk following ethanol administration

Alcohol and acetaldehyde were measured in milk and peripheral blood in chronic alcoholic rats, at 5 and 15 days of lactation. Ethanol in blood increased throughout lactation and the levels of acetaldehyde were much higher than in nonlactating alcoholic rats. The concentration of acetaldehyde in milk was always ca. 50% of that in blood, whereas that of ethanol varied within the range of 44-80% of the blood levels. Blood alcohol levels in the corresponding sucking pups were much lower than in maternal blood and increased throughout lactation. The time course of ethanol and acetaldehyde concentration in blood and milk were determined in normal lactating rats after cyanamide (40 mg/kg) and ethanol administration (2 or 4 g/kg). Milk alcohol reached higher concentrations than in blood within the first hour of ethanol administration, decreasing and remaining constant thereafter at ca. 65% of those in blood. Acetaldehyde levels in milk were always 35-45% lower than in blood. No alcohol dehydrogenase activity was found in homogenates of mammary tissue; however there was some aldehyde dehydrogenase activity. A significant decrease in mammary tissue aldehyde dehydrogenase was found in chronic alcoholic rats. The role of this enzyme is discussed.     

Effects of ADH2 overexpression in Saccharomyces bayanus during alcoholic fermentation

The effect of overexpression of the gene ADH2 on metabolic and biological activity in Saccharomyces bayanus V5 during alcoholic fermentation has been evaluated. This gene is known to encode alcohol dehydrogenase II (ADH II). During the biological aging of sherry wines, where yeasts have to grow on ethanol owing to the absence of glucose, this isoenzyme plays a prominent role by converting the ethanol into acetaldehyde and producing NADH in the process. Overexpression of the gene ADH2 during alcoholic fermentation has no effect on the proteomic profile or the net production of some metabolites associated with glycolysis and alcoholic fermentation such as ethanol, acetaldehyde, and glycerol. However, it affects indirectly glucose and ammonium uptakes, cell growth, and intracellular redox potential, which lead to an altered metabolome. The increased contents in acetoin, acetic acid, and L-proline present in the fermentation medium under these conditions can be ascribed to detoxification by removal of excess acetaldehyde and the need to restore and maintain the intracellular redox potential balance.     

Metabolic origin of acetaldehyde emitted by poplar (Populus tremula x (P. alba) trees

The metabolic origin and emission by the leaves of the tropospheric trace gas acetaldehyde were examined in 4-month-old poplar trees (Populus tremula x P. alba) cultivated under controlled environmental conditions in a greenhouse. Treatments which resulted in increased ethanol concentration of the xylem sap caused significantly enhanced rates of acetaldehyde and ethanol emission by the leaves. Leaves fed [¹⁴C]-ethanol via the transpiration stream emitted [^<14C]-acetaldehyde. These findings suggest that acetaldehyde in the leaves is synthesized by a metabolic pathway that operates in the opposite direction of alcoholic fermentation and results in oxidation of ethanol. Enzymatic studies showed that this pathway is mediated either by alcohol dehydrogenase (ADH; EC 1.1.1.1) or catalase (CAT; EC 1.11.1.6), both constitutively present in the leaves of poplar trees. Labelling experiments with [¹⁴C]-glucose indicated that the ethanol delivered to the leaves by the transpiration stream is produced in anaerobic zones of submersed roots by alcoholic fermentation. Anoxic conditions in the rhizosphere caused by flooding of the root system resulted in an activation of alcoholic fermentation and led to significantly increased ethanol concentrations in the xylem sap. These results support the hypothesis that acetaldehyde emitted by the leaves of trees is derived from xylem transported ethanol which is synthesized during alcoholic fermentation in the roots.Keywords: Acetaldehyde, emission, ethanol, anaerobiosis, Populus tremula x P. alba      

Acetaldehyde coenzyme A dehydrogenase of Escherichia coli.

Mutants of Escherichia coli (adh) in which alcohol dehydrogenase is derepressed under aerobic conditions were also found to overproduce acetaldehyde coenzyme a dehydrogenase. However, acetaldehyde coenzyme A dehydrogenase was induced by ethanol or acetaldehyde and subject to strong catabolite repression, whereas alcohol dehydrogenase was little affected by these conditions. Mutants no longer able to use ethanol as carbon source were isolated from an adh strain. Some of these mutants were revertants at the adh locus and no longer produced either alcohol dehydrogenase or acetaldehyde coenzyme A dehydrogenase. Others, designated acd, were found to lack only acetaldehyde coenzyme A dehydrogenase. The acd mutation was located at min 62 of the E. coli genetic map, the gene order being thyA-lysA-acd-serA-fda. Isolation of Tn10 insertions cotransducible with acd greatly simplified the mapping procedure.     

Evolutionary Changes in the Content of Dehydrogenase System Metabolites as a Mechanism of Adaptation to Cold in Higher Vertebrates

It has been found that the optimal body balance control under the conditions of the adaptation to cold is mostly determined by the ratio of the blood concentrations of endogenous ethanol and acetaldehyde related to the activities of liver alcohol dehydrogenase and aldehyde dehydrogenase in the order of increasing level of adaptation: higher vertebrates unadapted to cold, including human → small animals adapted to cold → large animals adapted to cold native to the North → hibernators, regardless of the species specificity and the environment.     

Effects of ALDH2 Genotype,PPI Treatment and L-Cysteine on Carcinogenic Acetaldehyde in Gastric Juice and Saliva after Intragastric Alcohol Administration

Acetaldehyde (ACH) associated with alcoholic beverages is Group 1 carcinogen to humans (IARC/WHO). Aldehyde dehydrogenase (ALDH2), a major ACH eliminating enzyme, is genetically deficient in 30–50% of Eastern Asians. In alcohol drinkers, ALDH2-deficiency is a well-known risk factor for upper aerodigestive tract cancers, i.e., head and neck cancer and esophageal cancer. However, there is only a limited evidence for stomach cancer. In this study we demonstrated for the first time that ALDH2 deficiency results in markedly increased exposure of the gastric mucosa to acetaldehyde after intragastric administration of alcohol. Our finding provides concrete evidence for a causal relationship between acetaldehyde and gastric carcinogenesis. A plausible explanation is the gastric first pass metabolism of ethanol. The gastric mucosa expresses alcohol dehydrogenase (ADH) enzymes catalyzing the oxidation of ethanol to acetaldehyde, especially at the high ethanol concentrations prevailing in the stomach after the consumption of alcoholic beverages. The gastric mucosa also possesses the acetaldehyde-eliminating ALDH2 enzyme. Due to decreased mucosal ALDH2 activity, the elimination of ethanol-derived acetaldehyde is decreased, which results in its accumulation in the gastric juice. We also demonstrate that ALDH2 deficiency, proton pump inhibitor (PPI) treatment, and L-cysteine cause independent changes in gastric juice and salivary acetaldehyde levels, indicating that intragastric acetaldehyde is locally regulated by gastric mucosal ADH and ALDH2 enzymes, and by oral microbes colonizing an achlorhydric stomach. Markedly elevated acetaldehyde levels were also found at low intragastric ethanol concentrations corresponding to the ethanol levels of many foodstuffs, beverages, and dairy products produced by fermentation. A capsule that slowly releases L-cysteine effectively eliminated acetaldehyde from the gastric juice of PPI-treated ALDH2-active and ALDH2-deficient subjects. These results provide entirely novel perspectives for the prevention of gastric cancer, especially in established risk groups.     

Enzymic analysis of the crabtree effect in glucose-limited chemostat cultures of Saccharomyces cerevisiae.

The physiology of Saccharomyces cerevisiae CBS 8066 was studied in glucose-limited chemostat cultures. Below a dilution rate of 0.30 h-1 glucose was completely respired, and biomass and CO2 were the only products formed. Above this dilution rate acetate and pyruvate appeared in the culture fluid, accompanied by disproportional increases in the rates of oxygen consumption and carbon dioxide production. This enhanced respiratory activity was accompanied by a drop in cell yield from 0.50 to 0.47 g (dry weight) g of glucose-1. At a dilution rate of 0.38 h-1 the culture reached its maximal oxidation capacity of 12 mmol of O2 g (dry weight)-1 h-1. A further increase in the dilution rate resulted in aerobic alcoholic fermentation in addition to respiration, accompanied by an additional decrease in cell yield from 0.47 to 0.16 g (dry weight) g of glucose-1. Since the high respiratory activity of the yeast at intermediary dilution rates would allow for full respiratory metabolism of glucose up to dilution rates close to mumax, we conclude that the occurrence of alcoholic fermentation is not primarily due to a limited respiratory capacity. Rather, organic acids produced by the organism may have an uncoupling effect on its respiration. As a result the respiratory activity is enhanced and reaches its maximum at a dilution rate of 0.38 h-1. An attempt was made to interpret the dilution rate-dependent formation of ethanol and acetate in glucose-limited chemostat cultures of S. cerevisiae CBS 8066 as an effect of overflow metabolism at the pyruvate level. Therefore, the activities of pyruvate decarboxylase, NAD+- and NADP+-dependent acetaldehyde dehydrogenases, acetyl coenzyme A (acetyl-CoA) synthetase, and alcohol dehydrogenase were determined in extracts of cells grown at various dilution rates. From the enzyme profiles, substrate affinities, and calculated intracellular pyruvate concentrations, the following conclusions were drawn with respect to product formation of cells growing under glucose limitation. (i) Pyruvate decarboxylase, the key enzyme of alcoholic fermentation, probably already is operative under conditions in which alcoholic fermentation is absent. The acetaldehyde produced by the enzyme is then oxidized via acetaldehyde dehydrogenases and acetyl-CoA synthetase. The acetyl-CoA thus formed is further oxidized in the mitochondria. (ii) Acetate formation results from insufficient activity of acetyl-CoA synthetase, required for the complete oxidation of acetate. Ethanol formation results from insufficient activity of acetaldehyde dehydrogenases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enzymic analysis of the crabtree effect in glucose-limited chemostat cultures of Saccharomyces cerevisiae

The physiology of Saccharomyces cerevisiae CBS 8066 was studied in glucose-limited chemostat cultures. Below a dilution rate of 0.30 h-1 glucose was completely respired, and biomass and CO2 were the only products formed. Above this dilution rate acetate and pyruvate appeared in the culture fluid, accompanied by disproportional increases in the rates of oxygen consumption and carbon dioxide production. This enhanced respiratory activity was accompanied by a drop in cell yield from 0.50 to 0.47 g (dry weight) g of glucose-1. At a dilution rate of 0.38 h-1 the culture reached its maximal oxidation capacity of 12 mmol of O2 g (dry weight)-1 h-1. A further increase in the dilution rate resulted in aerobic alcoholic fermentation in addition to respiration, accompanied by an additional decrease in cell yield from 0.47 to 0.16 g (dry weight) g of glucose-1. Since the high respiratory activity of the yeast at intermediary dilution rates would allow for full respiratory metabolism of glucose up to dilution rates close to mumax, we conclude that the occurrence of alcoholic fermentation is not primarily due to a limited respiratory capacity. Rather, organic acids produced by the organism may have an uncoupling effect on its respiration. As a result the respiratory activity is enhanced and reaches its maximum at a dilution rate of 0.38 h-1. An attempt was made to interpret the dilution rate-dependent formation of ethanol and acetate in glucose-limited chemostat cultures of S. cerevisiae CBS 8066 as an effect of overflow metabolism at the pyruvate level. Therefore, the activities of pyruvate decarboxylase, NAD+- and NADP+-dependent acetaldehyde dehydrogenases, acetyl coenzyme A (acetyl-CoA) synthetase, and alcohol dehydrogenase were determined in extracts of cells grown at various dilution rates. From the enzyme profiles, substrate affinities, and calculated intracellular pyruvate concentrations, the following conclusions were drawn with respect to product formation of cells growing under glucose limitation. (i) Pyruvate decarboxylase, the key enzyme of alcoholic fermentation, probably already is operative under conditions in which alcoholic fermentation is absent. The acetaldehyde produced by the enzyme is then oxidized via acetaldehyde dehydrogenases and acetyl-CoA synthetase. The acetyl-CoA thus formed is further oxidized in the mitochondria. (ii) Acetate formation results from insufficient activity of acetyl-CoA synthetase, required for the complete oxidation of acetate. Ethanol formation results from insufficient activity of acetaldehyde dehydrogenases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alcohol-induced autophagy contributes to loss in skeletal muscle mass

Patients with alcoholic cirrhosis and hepatitis have severe muscle loss. Since ethanol impairs skeletal muscle protein synthesis but does not increase ubiquitin proteasome-mediated proteolysis, we investigated whether alcohol-induced autophagy contributes to muscle loss. Autophagy induction was studied in: A) Human skeletal muscle biopsies from alcoholic cirrhotics and controls, B) Gastrocnemius muscle from ethanol and pair-fed mice, and C) Ethanol-exposed murine C2C12 myotubes, by examining the expression of autophagy markers assessed by immunoblotting and real-time PCR. Expression of autophagy genes and markers were increased in skeletal muscle from humans and ethanol-fed mice, and in myotubes following ethanol exposure. Importantly, pulse-chase experiments showed suppression of myotube proteolysis upon ethanol-treatment with the autophagy inhibitor, 3-methyladenine (3MA) and not by MG132, a proteasome inhibitor. Correspondingly, ethanol-treated C2C12 myotubes stably expressing GFP-LC3B showed increased autophagy flux as measured by accumulation of GFP-LC3B vesicles with confocal microscopy. The ethanol-induced increase in LC3B lipidation was reversed upon knockdown of Atg7, a critical autophagy gene and was associated with reversal of the ethanol-induced decrease in myotube diameter. Consistently, CT image analysis of muscle area in alcoholic cirrhotics was significantly reduced compared with control subjects. In order to determine whether ethanol per se or its metabolic product, acetaldehyde, stimulates autophagy, C2C12 myotubes were treated with ethanol in the presence of the alcohol dehydrogenase inhibitor (4-methylpyrazole) or the acetaldehyde dehydrogenase inhibitor (cyanamide). LC3B lipidation increased with acetaldehyde treatment and increased further with the addition of cyanamide. We conclude that muscle autophagy is increased by ethanol exposure and contributes to sarcopenia.     

Carbon isotope analysis of acetaldehyde emitted from leaves following mechanical stress and anoxia

Although the emission of acetaldehyde from plants into the atmosphere following biotic and abiotic stresses may significantly impact air quality and climate, its metabolic origin(s) remains uncertain. We investigated the pathway(s) responsible for the production of acetaldehyde in plants by studying variations in the stable carbon isotope composition of acetaldehyde emitted during leaf anoxia or following mechanical stress. Under an anoxic environment, C3 leaves produced acetaldehyde during ethanolic fermentation with a similar carbon isotopic composition to C3 bulk biomass. In contrast, the initial emission burst following mechanical wounding was 5–12‰ more depleted in ¹³C than emissions under anoxia. Due to a large kinetic isotope effect during pyruvate decarboxylation catalysed by pyruvate dehydrogenase, acetyl-CoA and its biosynthetic products such as fatty acids are also depleted in ¹³C relative to bulk biomass. It is well known that leaf wounding stimulates the release of large quantities of fatty acids from membranes, as well as the accumulation of reactive oxygen species (ROS). We suggest that, following leaf wounding, acetaldehyde depleted in ¹³C is produced from fatty acid peroxidation reactions initiated by the accumulation of ROS. However, a variety of other pathways could also explain our results, including the conversion of acetyl-CoA to acetaldehyde by the esterase activity of aldehyde dehydrogenase.     

Differences in hepatic and metabolic changes after acute and chronic alcohol consumption.

Hepatic metabolism of ethanol to acetaldehyde by the alcohol dehydrogenase pathway is associated with the generation of reducing equivalents as NADH. Conversely, reducing equivalents are consumed when ethanol oxidation is catalyzed by the NADPH dependent microsomal ethanol oxidizing system. Since the major fraction of ethanol metabolism proceeds via alcohol dehydrogenase and since the oxidation of acetaldehyde also generates NADH, an excess of reducing equivalents is produced. This explains a variety of effects following acute ethanol administration, including hyperlactacidemia, hyperuricemia, enhanced lipogenesis and depressed lipid oxidation. To the extent that ethanol is oxidized by the alternate microsomal ethanol oxidizing system pathway, it slows the metabolism of other microsomal substrates. Following chronic ethanol consumption, adaptive microsomal changes prevail, which include enhanced ethanol and drug metabolism, and increased lipoprotein production. Severe hepatic lesions (alcoholic hepatitis and cirrhosis) develop after prolonged ethanol consumption in baboons. These injurious alterations are not prevented by nutritionally adequate diets and can therefore be ascribed to ethanol rather than to dietary inadequacy.     

Alcohol ingestion,liver glutathione and lipoperoxidation: Metabolic interrelations and pathological implications

Data reviewed here indicate that acute and chronic ethanol ingestion induce a decrease in the concentration of GSH and an increase in lipoperoxidation in the liver both in experimental animals and in man, changes that are closely interrelated GSH depletion is suggested to be due to an oxidation in the liver tissue and to a translocation into the extrahepatic medium as free glutathione and/or as conjugates with ethanol-derived acetaldehyde. As a result, the hepatic GSH/GSSG ratio is drastically reduced. Lipoperoxidation seems to be related to the metabolism of ethanol and acetaldehyde by secondary pathways that are known to generate oxygen-related free radicals. Being lipoperoxidation a process associated with cell damage and death, its stimulation by ethanol ingestion could play a role in the production of alcoholic liver damage in man. The involvement of several contributory factors in the development of a high lipoperoxidative index in the liver in this situation is discussed.     

Acetaldehyde does not inhibit glutathione peroxidase and glutathione reductase from mouse liver in vitro

Acetaldehyde, the primary ethanol metabolite, has been implicated in the pathogenesis of alcoholic liver disease, but the mechanism involved is still under investigation. This study aims at the search for direct in vitro effects of different concentrations of acetaldehyde (30, 100 and 300microM) on the activities of glutathione reductase (GR), glutathione peroxidase (GPx) from liver supernatants, and the thiol-peroxidase activity of ebselen. They did not change after pre-incubation with acetaldehyde, which suggests that acetaldehyde does not have any direct effect. Nor were direct effects of acetaldehyde toward thiols, such as dithioerythritol and glutathione (GSH), observed either, even though GSH - measured as non-protein thiols from liver supernatants - were oxidized in the presence of acetaldehyde. In addition, acetaldehyde (up to 300microM) significantly oxidized GSH when incubated in the presence of commercially available gamma-glutamyltranspeptidase (GGT), but not in the presence of glutathione-S-transferase. The interaction between ebselen and GSH was also evaluated in an attempt to better understand the possible link between acetaldehyde and nucleophilic selenol groups. The formation and stability of ebselen intermediaries, produced in the chemical interaction between GSH and ebselen, were not affected by acetaldehyde either. Overall, the acetaldehyde oxidation of hepatic low-molecular thiols depends on mouse liver constituents and GGT is proposed as an important enzyme involved in this phenomenon. Thiol depletion, a phenomenon usually observed in the livers of alcoholic patients, can be related to GSH metabolism, and the involvement of GGT may reflect a molecular mechanism involved in thiol oxidation.     

Diurnal pattern of acetaldehyde emission by flooded poplar trees

The emission of the tropospheric trace gas acetaldehyde was determined in leaves of 4-month-old poplar trees ( Populus tremula × P. alba ) grown under controlled environmental conditions in a greenhouse. Using a dynamic cuvette system together with a high sensitivity laser-based photoacoustic detection unit, rates of acetaldehyde emission were measured with the high time resolution of about 15 min. Submergence of the roots resulted in the emission of acetaldehyde by the leaves. The emission increased linearly before reaching more or less steady-state values (ca 350 nmol m⁻² min⁻¹; ca 470 ng g⁻¹ dry weight min⁻¹) after approximately 6 h. Prolonged flooding of poplar trees resulted in a clear diurnal rhythm of acetaldehyde emission. The emission rates decreased when the light was switched off in the evening and peaked in the morning after the light was turned on again. This pattern significantly correlated with diurnal rhythms of stomatal conductance, photosynthesis, transpiration and with the concentrations of ethanol, the assumed precursor of acetaldehyde, in the xylem sap of flooded poplar trees. It may be concluded that under conditions of diminished stomatal conductance, acetaldehyde emission declines because its diffusive flux is reduced. Alternatively, reduced transpiration may decrease ethanol transport from the roots to the shoots and appreciable amounts of the acetaldehyde precursor ethanol are lacking in the leaves. The present results support the view that acetaldehyde emitted by the leaves of plants is derived from ethanol produced by alcoholic fermentation in submerged roots and transported to the leaves with the transpiration stream.     

Inhibition of mitochondrial aldehyde dehydrogenase and acetaldehyde oxidation by the glutathione-depleting agents diethylmaleate and phorone

Experiments were carried out to study the effect of two commonly used glutathione-depleting agents, diethylmaleate and phorone, on the oxidation of acetaldehyde and the activity of aldehyde dehydrogenase. The oxidation of acetaldehyde by intact hepatocytes was inhibited when the cells were incubated with diethylmaleate. Washing and resuspending the cells in diethylmaleate-free medium afforded protection against the inhibition of acetaldehyde oxidation. The oxidation of acetaldehyde by isolated rat liver mitochondria as well as by disrupted mitochondria in the presence of excess NAD+ was inhibited by diethylmaleate or phorone, indicating inhibition of the low-Km aldehyde dehydrogenase. In addition, diethylmaleate inhibited oxidation of acetaldehyde by the high-Km cytosolic aldehyde dehydrogenase. Significant accumulation of acetaldehyde occurred when ethanol was oxidized by hepatocytes in the presence, but not in the absence, of diethylmaleate. Thus, diethylmaleate blocks the oxidation of added or metabolically generated acetaldehyde, analogous to results with other inhibitors of the low-Km aldehyde dehydrogenase such as cyanamide. These results suggest that caution should be used in interpreting the effects of diethylmaleate or phorone on metabolic reactions, especially those involving metabolism of aldehydes such as formaldehyde, because, in addition to depleting glutathione, these agents inhibit the low-Km aldehyde dehydrogenase.     

淹水对两种甜樱桃砧木根系无氧呼吸酶及发酵产物的影响

以美早/东北山樱桃、美早/马哈利为试材,研究了淹水过程中两种甜樱桃砧木生长根、褐色木质根中无氧呼吸酶——丙酮酸脱羧酶(PDC)、乙醇脱氢酶(ADH)和乳酸脱氢酶(LDH)活性及褐色木质根的发酵产物——乙醛、乙醇和乳酸含量变化,结果表明:两类根系PDC、LDH活性均呈先升后降趋势,ADH活性变化在生长根中亦先升后降,而在褐色木质根中为上升趋势,三种酶活性变化幅度表现为生长根大于褐色木质根;美早/东北山樱桃两类根系中ADH和LDH活性增加幅度大于美早/马哈利,PDC则相反;两种砧木褐色木质根乙醛、乙醇含量呈升高趋势,乳酸含量先升后降;最终美早/东北山樱桃褐色木质根中乙醛含量低于美早/马哈利,乙醇含量则相反,而乳酸含量前者较早达峰值且高于后者峰值。     

Aldehyde Dehydrogenases in Rat Brain. Subcellular Distribution and Properties

Abstract: Kinetic studies suggested the presence of several forms of NAD-dependent aldehyde dehydrogenase (ALDH) in rat brain. A subcellular distribution study showed that low- and high- K _m activities with acetaldehyde as well as the substrate-specific enzyme succinate semialdehyde dehydrogenase were located mainly in the mitochondrial compartment. The low- K _m activity was also present in the cytosol (<20%). The low- K _m activity in the homogenate was only 10–15% of the total activity with acetaldehyde as the substrate. Two K _m values were obtained with both acetaldehyde (0.2 and 2000 μ m ) and 3,4-dihydroxyphenylacetaldehyde (DOPAL) (0.3 and 31 μ m ), and one K _m value with succinate semialdehyde (5 μ m ). The main part of the aldehyde dehydrogenase activities with acetaldehyde, DOPAL, and succinate semialdehyde, but only little activity of the marker enzyme for the outer membrane (monoamine oxidase, MAO), was released from a purified mitochondrial fraction subjected to sonication. Only small amounts of the ALDH activities were released from mitochondria subjected to swelling in a hypotonic buffer, whereas the main part of the marker enzyme for the intermembrane space (adenylate kinase) was released. These results indicate that the ALDH activities with acetaldehyde, DOPAL and succinate semialdehyde are located in the matrix compartment. The low- K _m activity with acetaldehyde and DOPAL, but not the high- K _m activities and succinate semialdehyde dehydrogenase, was markedly stimulated by Mg²⁺ and Ca²⁺ in phosphate buffer. The low- and high- K _m activities with acetaldehyde showed different pH optima in pyrophosphate buffer.     

Effects of Glycine on the Catecholamine Levels and Activities of Alcohol-Metabolizing Enzymes in Rats with Alcohol Intoxication and Addiction

We studied in rats the effects of peroral glycine introduction on the contents of catecholamines (CA) – noradrenaline (NA) and dopamine (DA) – in different brain structures (hypothalamus, midbrain, and neocortex), as well as the levels of adrenaline (A), NA, and DA in the blood and the activity of alcohol-metabolizing (AlM) enzymes – alcohol dehydrogenase (AlDH) and aldehyde dehydrogenase (AdhDH) – in the blood serum. The experimental group included animals with a disposition to alcohol consumption under conditions of free choice for drinking between an alcohol solution and water. The measurements were performed in animals in the state of acute alcohol intoxication (i.p. injection of 4 g/kg ethanol) or chronic alcohol addiction (formed due to a 3-month-long free access to ethanol solution). Introduction of 150 mg/kg glycine increased the NA and DA contents (the latter, to a lesser extent) in all examined brain structures; the NA level in the blood increased, while that of DA decreased. Under conditions of acute alcohol intoxication and chronic alcohol addiction, the ratio of the activities of AlM enzymes, AdhDH/AlDH, was significantly shifted toward values indicative of accumulation of acetaldehyde (AcAdh) in the tissues. This was accompanied by noticeable modifications of the CA contents in the brain structures and blood of the rats; in particular, the [DA]/[NA] ratio in the brain significantly increased. Introduction of glycine under conditions of acute alcohol intoxication provided normalization of the AdhDH/AlDH activity ratio. Obvious trends toward normalization of the CA levels in the brain structures were also observed in both acute and chronic experiments. In the latter case, the glycine treatment course resulted in a drop in the daily alcohol consumption by the animals. We conclude that glycine, which binds AcAdh and modifies the metabolism of CA transmitters, exerts a significant corrective influence on the pathogenetic mechanisms of alcohol addiction. Our experimental findings demonstrate that there are contact points between the acetaldehyde and catecholamine hypotheses of pathogenesis of alcoholism.     

Application of the reverse dept polarization-transfer pulse sequence to monitor in vitro and in vivo metabolism of 13C-ethanol by 1H-NMR spectroscopy

Using the reverse 13C----1H DEPT polarization-transfer pulse sequence the metabolism of 13C ethanol in vitro and in vivo has been monitored by 1H-NMR spectroscopy. Using yeast alcohol dehydrogenase, acetaldehyde, the hydrated form of acetaldehyde and acetate were identified as metabolites of [2-13C]-ethanol. The ratio of hydrated to free acetaldehyde was dependent upon the protein concentration of the reaction mixture. Binding of acetaldehyde in an irreversible Schiffs base resulted in optimal enzyme activity. Hepatocytes from rats fasted for 20 h, metabolised [1-13C] and [2-13C]ethanol in a linear fashion, but no [13C]acetaldehyde was detected. Metabolic integrity of the hepatocytes was confirmed with [2-13C]acetate. The addition of disulfiram (50 micron) to hepatocyte suspensions which had been incubated with [1-13C]ethanol, resulted in the resynthesis of [13C]ethanol. The amount of [13C]ethanol resynthesized under these conditions represents intracellular acetaldehyde whose concentration was in the range of 400-800 mumol/g wet weight of hepatocytes when 50 mM ethanol had been originally incubated with the hepatocyte suspension. These studies show how NMR-polarization transfer pulse sequences can be used to monitor the metabolism of 13C-ethanol in vivo, and provide a unique tool to measure in vivo concentrations of acetaldehyde. The studies also suggest that cytoplasmic aldehyde dehydrogenase may play a major role in hepatic ethanol metabolism.