Activities of human alcohol dehydrogenases in the metabolic pathways of ethanol and serotonin.

Alcohols and aldehydes in the metabolic pathways of ethanol and serotonin are substrates for alcohol dehydrogenases (ADH) of class I and II. In addition to the reversible alcohol oxidation/aldehyde reduction, these enzymes catalyse aldehyde oxidation. Class-I gammagamma ADH catalyses the dismutation of both acetaldehyde and 5-hydroxyindole-3-acetaldehyde (5-HIAL) into their corresponding alcohols and carboxylic acids.

A new HPLC-based method for the quantitative analysis of inner stratum corneum lipids with special reference to the free fatty acid fraction.

The inner stratum corneum is likely to represent the location of the intact skin barrier, unperturbed by degradation processes. In our studies of the physical skin barrier a new high-performance liquid chromatography (HPLC)-based method was developed for the quantitative analysis of lipids of the inner stratum corneum. All main lipid classes were separated and quantitated by HPLC/light scattering detection (LSD) and the free fatty acid fraction was further analysed by gas-liquid chromatography (GLC).

Coupling of ethanol metabolism to lipid biosynthesis: labelling of the glycerol moieties of sn-glycerol-3-phosphate, a phosphatidic acid and a phosphatidylcholine in liver of rats given [1,1-2H2]ethanol.

The mechanism behind ethanol-induced fatty liver was investigated by administration of [1,1-2H2]ethanol to rats and analysis of intermediates in lipid biosynthesis. Phosphatidic acid and phosphatidylcholine were isolated by chromatography on a lipophilic anion exchanger and molecular species were isolated by high-performance liquid chromatography in a non-aqueous system. The glycerol moieties of palmitoyl-linoleoylphosphatidic acid, the corresponding phosphatidylcholine and free sn-glycerol-3-phosphate were analysed by GC/MS of methyl ester t-butyldimethylsilyl derivatives.

Aldehyde dismutase activity of human liver alcohol dehydrogenase.

Human alcohol dehydrogenases of class I and class II but not class III catalyse NAD+-dependent aldehyde oxidation in addition to the NADH-dependent aldehyde reduction. The two reactions are coupled, i.e.

Metabolic profiles of steroids in urine of alcoholics after withdrawal.

The metabolic profiles of steroids in urine were analyzed in 13 male alcoholics during long-term abstinence, in most cases exceeding 3 months. The ratios of 5 beta- to 5 alpha-reduced steroid metabolites (etiocholanolone/androsterone and tetrahydrocortisol/allotetrahydrocortisol) were initially elevated but decreased slowly following withdrawal. The half-life of this normalization exceeded 3 weeks.

Inhibition of aldehyde dehydrogenases by methylene blue.

The effect of the redox dye methylene blue on the stability of NADH and on the activity of the enzyme aldehyde dehydrogenase (ALDH; EC 1.2.1.

Dehydrogenase-dependent metabolism of alcohols in gastric mucosa of deer mice lacking hepatic alcohol dehydrogenase.

Deer mice (Peromyscus maniculatus) lacking hepatic alcohol dehydrogenase (ADH) have been used as a model for studies of ethanol elimination catalysed by non-ADH systems like catalase and cytochrome P450. However, in an in vivo study on these animals (ADH- deer mice), we detected reversibility in the oxidation of [2H]ethanol, indicating that a major part of the ethanol elimination was due to a dehydrogenase (Norsten et al., J Biol Chem 264: 5593-5597, 1989).

Ethanol metabolism in isolated hepatocytes. Effects of methylene blue, cyanamide and penicillamine on the redox state of the bound coenzyme and on the substrate exchange at alcohol dehydrogenase.

Ethanol metabolism in hepatocytes increases the NADH/NAD+ ratio. The mechanism was investigated by measurements of the redox state of the coenzyme bound to alcohol dehydrogenase and of ethanol-acetaldehyde exchange and concomitant hydrogen transfer between ethanol molecules. Isolated hepatocytes from fed rats were incubated with cyclohexanone and cyclohexanol or with [1,1-2H2]-and [2,2,2-2H3]ethanol, followed by gas chromatographic determination of the redox state and isotope analysis of the ethanol by gas chromatography-mass spectrometry, respectively.

Decreased content of arachidonoyl species of phosphatidylinositol phosphates in pancreas of rats fed on an ethanol-containing diet.

Phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-diphosphate [PtdIns(4,5)P2] were isolated from the pancreas of rats fed an ethanol-containing liquid diet for 24 days and from the corresponding pair-fed controls. The isolation involved chromatography on a lipophilic anion exchanger in the phosphate form. The species composition was determined by fast-atom bombardment mass spectrometry.

Transfer of deuterium from [1,1-2H2]ethanol to steroids and organic acids in the rat testis.

Rats were given [1,1-2H2]ethanol in a single dose, and the 2H content was determined in testicular steroids and in organic acids of low molecular mass in the testis, liver and blood. The acids were quantified by capillary gas chromatography/mass spectrometry of t-butyldimethylsilyl derivatives with [2H4]lactate as internal standard. In addition to lactate, pyruvate, 3-hydroxybutyrate and acids of the tricarboxylic acid cycle, the testis was shown to contain 2-hydroxybutyrate, 2-hydroxy-2-methylbutyrate, 2-hydroxyisohexanoate and glycerate.

Oxidoreduction of butanol in deermice (Peromyscus maniculatus) lacking hepatic cytosolic alcohol dehydrogenase.

In view of conflicting information in the literature regarding enzyme systems responsible for alcohol oxidation in deermice previously reported to lack hepatic alcohol dehydrogenase (ADH) activity, the reversibility of butanol oxidation was studied in vivo and in liver-perfusion systems. Mixtures of [1,1-2H2]ethanol and butanol were given intraperitoneally to deermice lacking (ADH-) or possessing (ADH+) ADH activity, followed by analysis of alcohols in blood by GC/MS. 2H exchange between the two alcohols was seen in all experiments.

Acetaldehyde as a substrate for ethanol-inducible cytochrome P450 (CYP2E1).

Liver microsomes from starved and acetone-treated rats catalyzed NADPH-supported metabolism of acetaldehyde at a rate 8-fold higher than corresponding control microsomes; the Vmax was about 6 nmol/mg microsomal protein/min and the apparent Km 30 microM. The reaction was efficiently inhibited by anti-CYP2E1 IgG, but not by control IgG. Reconstituted membranes containing rat CYP2E1 and cytochrome b5 metabolized acetaldehyde with a Vmax of 20 nmol/nmol/min and an apparent Km of 30 microM, whereas CYP2B4 containing vesicles or vesicles without b5 were ineffective.

Stable isotope studies on steroid metabolism and kinetics: sulfates of 3 alpha-hydroxy-5 alpha-pregnane derivatives in human pregnancy.

The metabolism and production rates of 3 alpha-hydroxy-5 alpha-pregnan-20-one sulfate and the 3-sulfate and 3,20-disulfate of 5 alpha-pregnane-3 alpha,20 alpha-diol in pregnant women were studied. The steroid sulfates were labeled with deuterium in the 3 beta,11,11- or 3 beta,11,11,20 beta-positions and were injected intravenously. The deuterium content of steroids in the monosulfate and disulfate fraction of plasma collected at different times after the injection was determined by capillary column gas chromatography/mass spectrometry.

Analysis of aldehydic lipid peroxidation products in rat liver and hepatocytes by gas chromatography and mass spectrometry of the oxime-tert-butyldimethylsilyl derivatives.

A method for analysis of aliphatic aldehydes in biological samples is described. Cyclohexanone is added as internal standard and the samples are treated with hydroxylamine and perchloric acid. The oximes are extracted and converted to the oxime-tert-butyldimethylsilyl derivatives, which are quantitated by capillary gas chromatography and identified by mass spectrometry.

Compartmentation of acetyl CoA studied by analysis of tricarboxylic acid cycle acids and 3-hydroxybutyrate in bile of rats given [2,2,2-2H3]ethanol.

Acetate, 3-hydroxybutyrate, pyruvate, lactate, citrate, 2-oxoglutarate, succinate, fumarate and malate were analysed in rat bile by gas chromatography and gas chromatography/mass spectrometry of their O-melthyloxime-t-butyldimethylsilyl derivatives. The concentration of acetate increased to about 1.8 mmol/l after administration of [2,2,2-2H3]ethanol.

Concentration and turnover of estradiol in the rat uterus in vivo.

The concentrations and turnover of estradiol isolated from cytosolic and nuclear fractions of uteri from ovariectomized rats given estradiol, either in single injections or in continuous infusion, were analyzed by gas chromatography-mass spectrometry. The analytical method was validated for different organs and lower limits of analysis were established. After infusion of 20 ng x h-1 for 18-22 h, mean estradiol levels were 2.

Dehydrogenase-dependent ethanol metabolism in deer mice (Peromyscus maniculatus) lacking cytosolic alcohol dehydrogenase. Reversibility and isotope effects in vivo and in subcellular fractions.

Elimination of [2H]ethanol in vivo as studied by gas chromatography/mass spectrometry occurred at about half the rate in deer mice reported to lack alcohol dehydrogenase (ADH-) compared with ADH+ deer mice and exhibited kinetic isotope effects on Vmax and Km (D(V/K] of 2.2 +/- 0.1 and 3.

Mechanism and regulation of ethanol elimination in humans: intermolecular hydrogen transfer and oxidoreduction in vivo.

Ethanol metabolism was studied in four healthy volunteers by intravenous infusion of a mixture of [1,1-2H2]ethanol (1.0 mmol/kg) and [2,2,2-2H3]ethanol (1.0 mmol/kg) followed by blood sampling at 10-min intervals.

Influence of dietary fats on pancreatic phospholipids of chronically ethanol-treated rats.

Male rats were given liquid diets by pair-feeding for 24-30 days, and phosphatidylinositols in pancreas were analyzed as derivatives of diacylglycerols and fatty acids. Addition of arachidonic acid or changing the fat component (35 energy %) in the liquid diet from olive oil/corn oil to oil from Borago officinalis, which contains 22% gamma-linolenic acid, increased the fraction of arachidonoyl-containing species. This fraction was decreased by more than 50% by substituting ethanol for 36 of the 47 energy% provided by carbohydrate.

Hydrogen exchange during oxidoreduction and epimerization at C-3 of C19 steroid sulphates in the rat.

Mixtures of 17 beta-hydroxy-5 alpha-[16,16,17 alpha-2H3]androstan-3-one 17-sulphate and 5 alpha-[3 beta (or 3 alpha)-2H]androstane-3 alpha (or 3 beta), 17 beta-diol 17-sulphate were incubated with isolated hepatocytes from female rats or infused intravenously in female rats with bile fistulas. The androstanediols formed were analyzed by gas chromatography-mass spectrometry. Metabolism of 3H-labelled steroids was also studied in corresponding experiments.

Effect of ethanol on the redox state of the coenzyme bound to alcohol dehydrogenase studied in isolated hepatocytes.

Hepatocytes were isolated from fed female rats and incubated with a redox indicator system consisting of cyclohexanone and unlabelled or perdeuterated cyclohexanol. The concentrations and deuterium contents of these were measured by g.l.

Cytochrome P-450 dependent ethanol oxidation. Kinetic isotope effects and absence of stereoselectivity.

Deuterium isotope effects [D(V/K)] and stereoselectivity of ethanol oxidation in cytochrome P-450 containing systems and in the xanthine-xanthine oxidase system were compared with those of yeast alcohol dehydrogenase. The isotope effects were determined by using both a noncompetitive method, including incubation of unlabeled or [1,1-2H2]ethanol at various concentrations, and a competitive method, where 1:1 mixtures of [1-13C]- and [2H6]ethanol or [2,2,2-2H3]- and [1,1-2H2]ethanol were incubated and the acetaldehyde formed was analyzed by gas chromatography/mass spectrometry. The D(V/K) isotope effects of the cytochrome P-450 dependent ethanol oxidation were about 4 with liver microsomes from imidazole-, phenobarbital- or acetone-treated rabbits or with microsomes from acetone- or ethanol-treated rats.

Composition of platelet phosphatidylinositol and phosphatidylcholine after ethanol withdrawal.

Platelet phosphatidylinositol and phosphatidylcholine composition, ADP-induced platelet aggregation and associated thromboxane B2 formation were studied in alcoholics after a period of heavy drinking and in healthy non-alcoholic volunteers. The composition of these phospholipids in alcoholics was different from that seen in the control subjects. The most prominent change was a decrease in the relative amount of stearoyl-arachidonoyl species in the phosphatidylinositol fraction.