Effects of ethanol metabolism on oxidoreduction and intermolecular hydrogen transfer at C-17 in steroid 3-sulphates in vivo.

Steroid sulphates were infused intravenously in female rats, and metabolites were isolated from bile. Infused 3 beta-hydroxy-5 alpha-androstan-17-one 3-sulphate was excreted together with 5 alpha-androstane-3 beta,17 beta-diol disulphate, which formed a large part after ethanol administration. Results from infusions of the 3-sulphates of 5 alpha-[17 alpha-2H]androstane-2 beta,17 beta-diol and 3 beta-hydroxy-5 alpha-[2,2,4,4-2H4]androstan-17-one indicated that ethanol decreased the extent of transfer of the 17 alpha-deuterium and increased the reduction of 17-oxosteroid without affecting the oxidation of 17 beta-hydroxysteroid. Ethanol metabolism decreased the deuterium transfer from [17 alpha-2H]estradiol 3-sulphate to C-17 of 3 beta-hydroxy-5 alpha-androstan-17-one 3-sulphate. The results indicate that NADH from ethanol metabolism increased the concentration of oxidoreductase-NADH complex without affecting the corresponding complex with NAD+. The effects of ethanol on steroid reduction were dependent on the initial redox state of the enzyme-coenzyme complex. This redox state was modified by substrates for the enzyme, indicating slow dissociation of the complex. Thus, ethanol metabolism may interfere with the interactions between steroid oxidoreductions.

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. The deuterium labelling in the glycerol moiety of the phosphatidic acid was 2-3-times higher than in free sn-glycerol-3-phosphate, indicating that a specific pool of sn-glycerol-3-phosphate was used for the synthesis of phosphatidic acid in liver. The results indicate that NADH formed during ethanol oxidation is used in the formation of a pool of sn-glycerol-3-phosphate that gives rise to triacylglycerol and possibly fatty liver.

Origin of biliary phosphatidylcholines studied by coenzyme labelling with [1,1-2H2]ethanol.

The labelling of individual molecular species of phosphatidylcholines in bile and liver was measured in bile fistula rats given [1,1-2H2]ethanol immediately after the cannulation of the bile duct. Corresponding species in liver and bile were labelled to the same extent, the deuterium excess in the glycerol moiety (at C-2) of biliary molecules with rapid turnover possibly being slightly higher in the bile than in liver. The labelling of different positions and the half-life times of different molecular species were about the same as previously found 48 h after the cannulation. The only exception was the 1-stearoyl-2-linoleoyl species, which had a half-life time 5-7 times longer immediately after operation than after 48 h of biliary drainage. The results support our previous conclusion that the molecular species of phosphatidylcholines in liver and bile represent the same, or very similar, pool(s) of molecules.

Effects of chronic ethanol ingestion on steroid profiles in the rat testis.

Testosterone, seven of its potential precursors, three of its metabolites and estradiol were analyzed in testes from rats given ethanol for 23 days in a nutritionally adequate liquid diet. The results were compared to those obtained with pair-fed control rats. The concentrations of pregnenolone, progesterone, 17-hydroxyprogesterone, androstenedione and testosterone were markedly lowered in four of the five rats given ethanol. The concentrations of the other 3 beta-hydroxy-delta 5 steroids and estradiol were unchanged, resulting in significantly increased ratios between 17-hydroxypregnenolone and 17-hydroxyprogesterone (P less than 0.025) and between androstenediol and testosterone (P less than 0.025) in the ethanol-treated rats. The results indicate that chronic ethanol administration reduces formation of testosterone by affecting a step prior to pregnenolone. There may also be an effect on the conversion of some 3 beta-hydroxy-delta 5 to the corresponding 3-oxo-delta 4 steroids. The levels of testosterone and three other steroids in testes of rats given the liquid diet were significantly lower than those in testes of animals fed a standard rat chow. This indicates a dietary influence on testicular steroid concentrations.

Exchange of methyl hydrogens in ethanol during incorporation in bile acids in vivo.

[2,2,2-2H]Ethanol was administered continuously to bile fistula rats for 72 h, with or without (--)-hydroxycitrate. The deuterium labelling of biliary bile acids was determined by GC-MS and 13C NMR. Difference spectra between 2H,1H- and 1H-decoupled 13C NMR spectra showed the presence of partly deuterated methyl and methylene groups in methyl cholate, indicating exchange of deuterium in [2,2,2-2H]ethanol for protium prior to or during incorporation of acetate into the bile acid. The extent of exchange was 20--30% as calculated from the isotopic composition of a fragment ion containing one methyl and one methylene group derived from C-2 of acetate. The exchange was unaffected by (--)-hydroxycitrate, indicating that it was not due to reversible incorporation of deuterated acetate into citrate. About 60% of the acetyl-CoA serving as precursor of cholic and chenodeoxycholic acids were derived from ethanol. This value was not changed by administration of (--)-hydroxycitrate. The half-life time of cholesterol molecules acting as precursors of both bile acids was about 50 h in the presence of (--)-hydroxycitrate, which is about the same as previously found in the absence of the inhibitor.

Biosynthesis of 5 alpha- and 5 beta-cholanoic acid derivatives during metabolism of [1,1-2H]- and [2,2,2-2H]ethanol in the rat.

Female bile fistula rats were given [1,1-2H]ethanol in a single dose or [2,2,2-2H]ethanol repeatedly for 24 h and incorporation of deuterium into the following bile acids was determined: taurine conjugates of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5(alpha and beta)-cholanoic, 3 alpha, 7 alpha-dihydroxy-5(alpha and beta)-cholanoic and 3 alpha, 6 beta, 7 alpha-trihydroxy-5 beta-cholanoic acids; sulphates of 3(alpha and beta), 7 alpha, 12 alpha-trihydroxy-5 alpha-cholanoic, and 3(alpha and beta), 7 alpha-dihydroxy-5 alpha-cholanoic acids. The kinetics of deuterium incorporation from [2,2,2-2H]ethanol was the same for all bile acids indicating that they were formed from a single pool of cholesterol. The labelling pattern of bile acids formed during metabolism of [1,1-2H]-ethanol indicated that the hydrogen at C-5 was labelled in all bile acids. Taken together with previous results this indicates that 3-oxo-4-cholenoic acid is not an intermediate in the formation of allo bile acids. The results support the view that formation of allo bile acids via a mitochondrial pathway is of little importance in the bile fistula rat.

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. the enzymes display dismutase activity. Dismutase activity of recombinantly expressed human class I isozymes beta1beta1 and gamma2gamma2, class II and class III alcohol dehydrogenases was assayed with butanal as substrate by gas chromatographic-mass spectrometric quantitations of butanol and butyric acid. The class I gamma2gamma2 isozyme showed a pronounced dismutase activity with a high kcat, 1300 min(-1), and a moderate Km, 1.2 mM. The class I beta1beta1 isozyme and the class II alcohol dehydrogenase showed moderate catalytic efficiencies for dismutase activity with lower kcat values, 60-75 min(-1). 4-Methylpyrazole, a potent class I ADH inhibitor, inhibited the class I dismutation completely, but cyanamide, an inhibitor of mitochondrial aldehyde dehydrogenase, did not affect the dismutation. The dismutase reaction might be important for metabolism of aldehydes during inhibition or lack of mitochondrial aldehyde dehydrogenase activity.

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. Cyanamide and methylene blue decreased the redox shift caused by ethanol and increased the rates of acetaldehyde reduction during the exchange. Both drugs increased the extent of hydrogen transfer between ethanol molecules during oxidoreduction. Penicillamine had no significant effect on the ethanol-induced change in redox state of the bound coenzyme although it decreased the rate of acetaldehyde reduction. The results indicate that methylene blue inhibits aldehyde dehydrogenase and that accumulation of acetaldehyde decreases the redox effects of ethanol. The redox effect appears to result primarily from rapid elimination of acetaldehyde and equilibration with the NAD system on the alcohol dehydrogenase, but is not enhanced by further decreases in acetaldehyde concentration. Thus, penicillamine could probably be used to decrease the concentration of acetaldehyde without increasing the redox effects.

Decrease in arachidonoyl-containing phosphatidylinositols in pancreas of rats fed an ethanol-containing diet.

The composition of the glycerophosphatides in pancreas and liver was studied in rats fed an ethanol-containing diet and in pair-fed controls. The fraction of arachidonoyl-containing phosphatidylinositols in pancreas was much lower in the former rats, also when the rats were starved for a final 24 hr period. This fraction was also lower in fed than in starved control rats. The effect was not observed after acute administration of ethanol. It is suggested that the decrease in arachidonoyl-containing phosphatidylinositols was due to chronic pancreatic hyperfunction in the ethanol-fed rats.

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 1.2.1.3) was examined. NADH was measured by HPLC with fluorometric or spectrophotometric detection. The ALDH activity assays were carried out by following the formation of 3,4-dihydroxyphenylacetic acid (DOPAC) from 3,4-dihydroxyphenylacetaldehyde (DOPAL) using HPLC and electrochemical detection. Incubation of NADH solutions in the presence of methylene blue resulted in a time-dependent direct oxidation of NADH. Methylene blue inhibited the human erythrocyte and leukocyte ALDHs and the rat liver mitochondrial low-Km ALDH in a concentration-dependent manner. The inactivation was reversible by dilution, and kinetic analysis indicated that methylene blue inhibits the rat liver mitochondrial low-Km and human erythrocyte ALDHs competitively with respect to DOPAL, while no effect of the NAD+ concentration was apparent. For the rat liver low-Km ALDH, a Ki of 8.4 +/- 2.8 microM (mean +/- SD; N = 5) was calculated. The inhibition of ALDH and the resulting decrease in the redox effect on the NAD system bound to alcohol dehydrogenase (EC 1.1.1.1) could explain the protective effect of methylene blue against metabolic redox effects of ethanol.

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). In the present investigation, we found significant ethanol oxidizing activity in the gastric mucosa of the deer mice. Reversibility was demonstrated by the use of [2H]acetaldehyde and gas chromatography-mass spectrometry of the products. The kinetic 2H isotope effect of the gastric system was about 3.0 and the system was comparatively insensitive to inhibition by 4-methylpyrazole. The behavior of the deer mice gastric ADH in isoelectric focusing and its higher activity with longer alcohols as substrates indicated similarity with the previously described human class IV enzymes. Our data are in agreement with results obtained in vivo and indicate that ethanol is oxidized extrahepatically in ADH- deer mice. This has to be taken into account when deer mice are used to study non-ADH-dependent ethanol oxidation in vivo.

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. The change was most marked in patients with signs of liver injury, and may reflect a relative decrease of the activity of hepatic 5 alpha-reductase. The ratio between cortisol metabolites carrying a 11 beta-hydroxy and an 11-oxo group was elevated in the patients and showed no tendency to normalize. This might reflect a decrease in the peripheral inactivation of cortisol.

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. Particularly this species of PI might be involved in the transmission of transmembrane signals. During detoxification changes were also observed in the extent of ADP-induced platelet aggregation and the amount of thromboxane B2 produced. Changes in platelet phospholipid composition might influence platelet reactivity in alcoholics.

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. The injected steroid sulfates underwent oxidoreduction at C-20 and 16 alpha-hydroxylation. In addition, the 3-sulfate of 5 alpha-pregnane-3 alpha,20 alpha-diol became hydroxylated at C-21. The pregnanediol and pregnanetriol monosulfates were also converted to disulfates. No evidence was obtained for a metabolic sequence involving hydrolysis, oxidoreduction, and resulfation at the C-3 position. Production rates and rates of metabolic transformations were determined using different one- and two-pool models. The production rate of the pregnanolone/pregnanediol monosulfate couple was 0.08 to 0.5 mmol/24 h, the variability probably depending both on individual factors and stage of pregnancy. The half-life time for oxidation and reduction at C-20 was 0.1 to 0.4 hours, reduction being the faster process. The half-life time for the turnover of the steroid skeleton was 1.3 to 3.3 hours. The injected steroid monosulfates were 16 alpha-hydroxylated at a rate of 1 to 8 mumol/24 h. A significant fraction of these 16 alpha-hydroxylated steroid sulfates, 0.5 to 25 mumol/24 h, was formed from other, probably unconjugated, precursors. The 16 alpha-hydroxylated steroid monosulfates underwent rapid oxidoreduction at C-20. The 3-sulfate of 5 alpha-pregnane-3 alpha,20 alpha-diol was hydroxylated at C-21. The production rate of 5 alpha-pregnane-3 alpha,20 alpha,21-triol 3-sulfate was 8 to 36 mumol/24 h in four women and 180 mumol/24 h in one woman, and this steroid was not formed from other precursors to a significant extent. 5 alpha-Pregnane-3 alpha,20 alpha-diol disulfate was a metabolic end product accounting for a major part of the elimination of the steroids injected. Its half-life time was 1.4 to 2.8 hours. The results show that the formation of sulfated steroids with a 3 alpha-hydroxy-5 alpha configuration may account for 50% of the metabolism of progesterone in late pregnancy.

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). Mass spectrometry (MS) was used for peak identification and flame ionization detection (FID) for quantitation. Special attention was paid to the free fatty acid fraction since unsaturated free fatty acids may exert a key function in the regulation of the skin barrier properties by shifting the physical equilibrium of the multilamellar lipid bilayer system towards a noncrystalline state. Our results indicated that the endogenous free fatty acid fraction of the stratum corneum barrier lipids in essence exclusively consisted of saturated long-chain free fatty acids. This fraction was characterized as a very stable population (low interindividual peak variation) dominated by saturated lignoceric acid (C24:0, 39 molar%) and hexacosanoic acid (C26:0, 23 molar%). In addition, trace amounts of very long-chain (C32-C36) saturated and monounsaturated free fatty acids were detected in human forearm inner stratum corneum. Our analysis method gives highly accurate and precise quantitative information on the relative composition of all major lipid species present in the skin barrier. Such data will eventually permit skin barrier model systems to be created which will allow a more detailed analysis of the physical nature of the human skin barrier.

NAD+-dependent ethanol oxidation: redox effects and rate limitation.

Effects of ethanol on interconversion of cyclohexanol and cyclohexanone was studied in isolated hepatocytes. Oxidation and reduction catalyzed by alcohol dehydrogenase were markedly inhibited and stimulated, respectively. The changed ratio between the rates indicated that the ratio of NAD+ to NADH bound to alcohol dehydrogenase decreased several hundred times. This is much more than for the NAD+ system used by, e.g., lactate dehydrogenase, and deuterium from [1,1-2H2] ethanol was incorporated in cyclohexanol much more than in, e.g., lactate. These results indicate that the coenzyme bound to alcohol dehydrogenase is not equilibrated with free coenzyme. Thus, the dissociation of NADH might be rate-limiting for ethanol oxidation. Deuterium transfer from chiral [1-2H] ethanols and [2-2H] glycerol in hepatocytes indicated that cytosolic malate dehydrogenase and lactate dehydrogenase were not completely equilibrated, whereas there was no difference in the utilization of NADH formed at alcohol dehydrogenase and at glycerol-3-phosphate dehydrogenase. Fluxes in redox reactions during ethanol oxidation may be too high for equilibration of cytosolic dehydrogenases.

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. A smaller difference between ethanol-fed and control rats was seen in the composition of phosphatidylcholines and phosphatidylethanolamines. There was no difference in the composition of phosphatidylinositols when fat, instead of ethanol, was used to substitute the 36 energy % in the diet containing olive oil/corn oil. Substituting ethanol for 28 of 35 energy% provided by fat as corn oil in a liquid diet had no effect on the fraction of arachidonoyl-containing species. The results indicate that the effect of ethanol on phosphatidylinositols in pancreas is not due to a deficiency of arachidonic acid, and that the effect of the ethanol-containing diet is not due to the lowered carbohydrate content. However, high contents of fat or of ethanol appear to be necessary for the effect.

Phosphatidylinositol composition in pancreas and submaxillary gland of ethanol-fed rats.

The possibility that changes in the stimulus-secretion coupling are involved in the etiology of pancreatitis was investigated by analysis of the molecular composition of the phosphatidylinositols in pancreas of rats fed a liquid ethanol-containing diet and pair-fed controls. The arachidonoyl-containing phosphatidylinositols were about half as abundant in the ethanol-fed rats. Opposite differences were seen for the major species containing linoleoyl, oleoyl or stearoyl groups at C-2. Ethanol-induced lipid peroxidation did not seem to be involved, since carbon tetrachloride administration had no effect on the composition. In the submaxillary gland, that has a similar stimulus-secretion coupling, the arachidonoyl-containing phosphatidylinositols constituted about a 25% smaller fraction in the ethanol-fed rats. Dexamethasone administration did not change the effect. Possibly, decreased secretory response in these glands in ethanol-fed rats is caused by the change in phosphatidylinositol composition.

Steroid profiles in urine and plasma of alcoholics during withdrawal.

Metabolic profiles of steroids in urine and plasma were analyzed in 14 male and four female alcoholics during withdrawal. The daily excretion of 30 conjugated steroids in urine and the concentration of 13 steroid sulfates in plasma were measured on days 1, 7 and 29 of the period of observation, which started on day 5-7 of abstinence. While the total excretion of cortisol metabolites was normal in most cases, the profiles of metabolites were changed in the alcoholics during the period of observation. The ratio between tetrahydrocortisol and tetrahydrocortisone exceeded the mean normal value by more than one standard deviation in 97% of the samples analyzed. The same was true of the ratio between 20-hydroxy and 20-oxosteroids in 90% of the samples. The differences between alcoholic and healthy subjects were statistically significant (p less than 0.001). The major change in plasma was a significantly increased concentration of 5-androstene-3 beta, 17 beta-diol disulfate on the first day of the study. The concentration decreased to normal values during the first month of withdrawal. The rate of excretion of this steroid in urine was increased in half of the patients and also decreased with time. The rate of excretion and the degree of fatty infiltration in liver biopsies were positively correlated. It is suggested that the ratios between cortisol metabolites in urine might be of value as biochemical markers in alcoholism, and that the absolute or relative concentrations of steroid disulfates in plasma might serve as an indicator of recent alcohol intake.