Metabolic aspects of alcoholic liver damage: 1984/5 update. 1. Epidemiology and alcohol metabolism

In western industrialized countries ethanol is an important etiologic factor in the development of cirrhosis of the liver. Metabolic, immunologic and physico-chemical alterations of the hepatocyte due to ethanol are involved in the pathogenesis of alcoholic liver disease. However, the mechanisms by which ethanol damages the liver are far from clear. During the last two decades, the effect of ethanol on multiple biochemical pathways of the hepatocyte has been investigated intensively. The present paper is focusing on the metabolic aspects of alcoholic liver disease. In the first part of the review, special emphasis has been led on the metabolites of ethanol oxidation, while in the second part microsomal enzyme induction due to alcohol has been discussed. More than 90% of ethanol metabolism takes place in the liver via cytoplasmic alcoholdehydrogenase (ADH) and via a microsomal ethanol oxidizing system (MEOS). The products of these reactions are reduced nicotinadenine dinucleotide phosphate (NADH), acetaldehyde and acetate. NADH alters the redox state of the liver cell favouring all reductive processes. This shift in metabolic pathways results in hyperlactacidaemia, lactacidosis, ketosis and hyperuricaemia. Disturbances of the carbohydrate metabolism may lead either to hypo- or hyperglycaemia. The altered redox state also influences the metabolic pathways of lipid metabolism leading to lipid accumulation within the hepatocyte which can be morphologically observed as alcoholic fatty liver. In addition, porphyrin and collagen metabolism is also affected by the increased NADH/NAD+ ratio. On the other hand, acetaldehyde damages the microtubular system and the mitochondria. Acetaldehyde may also be responsible for the increased lipidperoxidation after chronic ethanol ingestion.     

Acetaldehyde alone may initiate hepatocellular damage in acute alcoholic liver disease.

Acetaldehyde may be the injurious agent in acute alcoholic liver disease. It has been suggested that the mechanism of liver injury in this situation may be immunologically mediated. In the present study acetaldehyde has been bound to human liver plasma membranes. The activation of C3 by the acetaldehyde/membrane product was measured by immunofixation of the separated C3 components. Activation of C3 by acetaldehyde exposed liver plasma membranes was increased to 16.4% compared with 6% by non-exposed membranes (p = 0.004). Human liver plasma membranes bound 212 +/- 18 nmol acetaldehyde per mg membrane protein. The binding constant was 439 +/- 81 microM. It is concluded that acetaldehyde bound to human liver plasma membranes activates the complement sequence and this may be the initial stage in the pathogenesis of acute alcoholic liver disease.     

Role of Acetaldehyde in the Pathogenesis of Alcoholic Liver Disease

Acetaldehyde is the primary metabolic product of alcohol metabolism in the liver and is highly reactive. High concentrations may be achieved locally in the liver during alcohol abuse. Like alcohol, acetaldehyde appears not to be directly toxic to the liver cell but it binds non-enzymatically to free amino groups in the proteins of the liver cell. The product of this addition reaction, the adduct, can alter the surface charge and structural conformation of protein molecules to expose new antigenic sites. The highly variable susceptibility to alcoholic liver disease is compatible with an immunologically mediated liver damage. The acetaldehyde adduct is also pro inflammatory in its own right and will activate the complement sequence, recruit and cause degranulation of neutrophils and stimulate neutrophil superoxide anion production. Acetaldehyde is also a substrate for free radical production in the liver. There is increasing evidence of both free radical and immunologically mediated damage in alcoholic liver disease. Although there is a increasing body of evidence to suggest that acetaldehyde is capable of inducing liver damage by such mechanisms, there is as yet no evidence to confirm that this actually occurs in alcohol abuse and the role of acetaldehyde remains speculative.     

Effects of acetaldehyde on hepatocyte glycerol uptake and cell size: implication of aquaporin 9

Background: The effects of ethanol and acetaldehyde on uptake of glycerol and on cell size of hepatocytes and a role Aquaporin 9 (AQP9), a glycerol transport channel, were evaluated. Methods: The studies were done in primary rat and mouse hepatocytes. The uptake of [¹⁴C] glycerol was determined with hepatocytes in suspension. For determination of cell size, rat hepatocytes on coated dishes were incubated with a lipophilic fluorochrome that is incorporated into the cell membrane and examined by confocal microscopy. A three‐dimensional z scan of the cell was performed, and the middle slice of the z scan was used for area measurements. Results: Acute exposure to acetaldehyde, but not to ethanol, causes a rapid increase in the uptake of glycerol and an increase in hepatocyte size, which was inhibited by HgCl₂, an inhibitor of aquaporins. This was not observed in hepatocytes from AQP9 knockout mice, nor observed by direct application of acetaldehyde to AQP9 expressed in Xenopus Laevis oocytes. Prolonged 24‐hour exposure to either acetaldehyde or ethanol did not result in an increase in glycerol uptake by rat hepatocytes. Acetaldehyde decreased AQP9 mRNA and AQP9 protein, while ethanol decreased AQP9 mRNA but not AQP9 protein. Ethanol, but not acetaldehyde, increased the activities of glycerol kinase and phosphoenolpyruvate carboxykinase. Conclusions: The acute effects of acetaldehyde, while mediated by AQP9, are probably influenced by binding of acetaldehyde to hepatocyte membranes and changes in cell permeability. The effects of ethanol in enhancing glucose kinase, and phosphoenolpyruvate carboxykinase leading to increased formation of glycerol‐3‐phosphate most likely contribute to alcoholic fatty liver.     

Acetalde hyde-Induced Stimulation of Collagen Synthesis and Gene Expression Is Dependent on Conditions of Cell Culture: Studies with Rat Lipocytes and Fibroblasts

Acetaldehyde has been proposed as a mediator of fibrogenesis in alcoholic liver disease, based in part on its ability to stimulate collagen synthesis by hepatic lipocytes in late primary or passaged culture. In this study, we examined the effect of acetaldehyde on rat lipocytes and fibroblasts at various stages of culture, in an effort to determine whether culture-related events influence responsiveness to this compound. Lipocytes from normal rat liver were studied in primary culture at 3 and 7 days after plating; fibroblasts were studied in subculture, at subconfluent and confluent densities. Both cell types were incubated with 100 μM acetaldehyde for 24 hr followed by measurement of collagen synthesis and type I collagen gene expression. Acetaldehyde had no effect on lipocytes at either 3 or 7 days in primary culture. The inability of acetaldehyde to stimulate collagen synthesis in primary culture was not attributable to toxicity, because cell morphology and total protein synthesis were identical in both treated and untreated cultures. Fibroblasts exhibited a variable response to acetaldehyde that was dependent on cell density: subcon-fluent cells contained similar amounts of type I collagen mRNA in both the presence and absence of acetaldehyde, whereas confluent cells exhibited a 2- to 3-fold increase in collagen mRNA levels upon acetaldehyde exposure. To determine whether quiescent lipocytes would respond to acetaldehyde in a culture system that mimics the hepatic environment in vivo, lipocytes were plated in coculture with hepatocytes on a basement membrane gel and incubated with 20 mM ethanol for 72 hr. Direct communication between these two cell types did not provoke lipocyte activation, even in the setting of ethanol oxidation. We conclude from these experiments that acetaldehyde is not a primary stimulus to lipocyte activation in vivo. Acetaldehyde may enhance collagen synthesis by lipocytes, but its activity appears restricted to cells that have undergone a prior priming event in culture or in vivo. Of the many phenotypic changes that occur in lipocytes during the first week of primary culture, none sensitizes them to the fibrogenic effects of acetaldehyde.     

The detection of acetaldehyde/liver plasma membrane protein adduct formed in vivo by alcohol feeding

In order to assess whether acetaldehyde adducts with liver plasma membrane proteins are formed in vivo during alcohol ingestion, liver plasma membranes were prepared from control rats and rats fed on 10% ethanol from weaning and the amino acid constituents of liver plasma membrane proteins were assessed by reversed phase liquid chromatography of an acid hydrolysate of the membranes. The retention time of acetaldehyde/lysine adduct after stabilisation through reduction was determined by chromatography of an acid hydrolysate of polylysine pretreated with acetaldehyde. The presence of a peak with identical retention time to the acetaldehyde/lysine adduct was detected in liver plasma membranes isolated from alcohol-fed rats indicating adduct formation in vivo. The adduct was detectable only when the membranes were prepared by a rapid (Percoll) method, suggesting that the adduct may be unstable. The findings are consistent with the hypothesis that the inflammation of acute alcoholic liver disease may be initiated by the product of acetaldehyde/membrane binding in vivo.     

The Effects of Acetaldehyde and Disulfiram on Albumin Synthesis in the Isolated Perfused Rabbit Liver

Acetaldehyde infusions inhibit albumin synthesis in the liver from fed donors but not in the livers from fasted donors. The inhibition of acetaldehyde metabolism with 4-MP and disulfiram reverses this finding, suggesting that acetaldehyde per se is not the toxic agent. Disulfiram stimulates albumin synthesis in livers from fasted donors, and the presence of acetaldehyde does not prevent this process. The effects of ethanol infusions cannot be explained as due to the presence of acetaldehyde; some intermediate metabolic step may be the basis of the inhibition of albumin production and polysome disaggregation in the presence of ethanol.     

Effect of Acetaldehyde Upon Cathepsin G and Chymase. NRAS Implications

Hypertension is commonly observed in alcoholics. Both the renin-angiotensin system (RAS) and the non-renin-angiotensin system (NRAS) have been implicated in the dynamics of blood pressure maintenance. In bilaterally nephrectomized rats, acetaldehyde has been reported to enhance the generation of the rate-limiting angiotensin I (ANG I) in the plasma, and in humans it inhibits the activity of several angiotensinases (A, B, and M) in the serum, thereby promoting a hypertensive set of reactions. We report here the results of a study on the effect of acetaldehyde upon cathepsin G and mast cell chymase. Acetaldehyde enhanced cathepsin G activity at all of the concentrations tested between 11.2 and 223.5 mM in a statistically significant manner. Since cathepsin G is one of several enzymes transforming ANG I into ANG II and is also capable of cleaving ANG II directly from angiotensinogen, we suggest that alcoholism, which will generate exogenous acetaldehyde from ingested alcohol, may be a contributory factor for an elevated cathepsin G activity and, consequently, hypertension via the NRAS. Chymase activity also is elevated in the presence of 440 mM acetaldehyde and diminished in the presence of 27 mM acetaldehyde. Since both enzymes also degrade ANG II, the degradative effects of each enzyme on ANG II may neutralize one another.     

The Effect of Cyanamide on Acetaldehyde Oxidation by Isolated Rat Liver Mitochondria and on the Inhibition of Pyruvate Oxidation by Acetaldehyde

Compared to other substrates, the oxidation of pyruvate by isolated mitochondria is especially sensitive to inhibition by acetaldehyde. It is not known whether this inhibition represents a direct effect of acetaldehyde or requires the metabolism of acetaldehyde. Experiments were therefore carried out in the presence of cyanamide, an inhibitor of aldehyde dehydrogenase. After a brief incubation period, cyanamide inhibited the state 4 and state 3 rate of acetaldehyde (0.1–1.0 mM) oxidation by isolated rat liver mitochondria. Little inhibition was found in the absence of the incubation period. Maximum inhibition was found at cyanamide concentrations of 0.01 to 0.033 mM. Cyanamide also inhibited the activity of aldehyde dehydrogenase assayed in disrupted mitochondrial fractions. The inhibition by cyanamide was specific since cyanamide did not affect mitochondrial oxidation of succinate, glutamate, or pyruvate. Acetaldehyde inhibited the state 3 rate of pyruvate oxidation by liver mitochondria. Despite preventing acetaldehyde oxidation, cyanamide did not prevent the inhibition of pyruvate oxidation by acetaldehyde. These results indicate that (a) cyanamide can be used as an effective in vitro inhibitor of acetaldehyde oxidation and (b) the unique sensitivity of pyruvate oxidation to acetaldehyde represents a direct effect of acetaldehyde on pyruvate dehydrogenase.     

Hepatic, Metabolic and Toxic Effects of Ethanol: 1991 Update

Until two decades ago, dietary deficiencies were considered to be the only reason for alcoholics to develop liver disease. As the overall nutrition of the population improved, more emphasis was placed on secondary malnutrition and direct hepatotoxic effects of ethanol were established. Ethanol is hepatotoxic through redox changes produced by the NADH generated in its oxidation via the alcohol dehydrogenase pathway, which in turn affects the metabolism of lipids, carbohydrates, proteins, and purines. Ethanol is also oxidized in liver microsomes by an ethanol-inducible cytochrome P-450 (P-450IIE1) that contributes to ethanol metabolism and tolerance, and activates xenobiotics to toxic radicals thereby explaining increased vulnerability of the heavy drinker to industrial solvents, anesthetic agents, commonly prescribed drugs, over-the-counter analgesics, chemical carcinogens, and even nutritional factors such as vitamin A. In addition, ethanol depresses hepatic levels of vitamin A, even when administered with diets containing large amounts of the vitamin, reflecting, in part, accelerated microsomal degradation through newly discovered microsomal pathways of retinol metabolism, inducible by either ethanol or drug administration. The hepatic depletion of vitamin A is strikingly exacerbated when ethanol and other drugs were given together, mimicking a common clinical occurrence. Microsomal induction also results in increased production of acetaldehyde. Acetaldehyde, in turn, causes injury through the formation of protein adducts, resulting in antibody production, enzyme inactivation, decreased DNA repair, and alterations in microtubules, plasma membranes and mitochondria with a striking impairment of oxygen utilization. Acetaldehyde also causes glutathione depletion and lipid peroxidation, and stimulates hepatic collagen production by the vitamin A storing cells (lipocytes) and myofibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)     

An optimized method for the measurement of acetaldehyde by high-performance liquid chromatography

Background: Acetaldehyde is produced during ethanol metabolism predominantly in the liver by alcohol dehydrogenase and rapidly eliminated by oxidation to acetate via aldehyde dehydrogenase. Assessment of circulating acetaldehyde levels in biological matrices is performed by headspace gas chromatography and reverse phase high‐performance liquid chromatography (RP‐HPLC). Methods: We have developed an optimized method for the measurement of acetaldehyde by RP‐HPLC in hepatoma cell culture medium, blood, and plasma. After sample deproteinization, acetaldehyde was derivatized with 2,4‐dinitrophenylhydrazine (DNPH). The reaction was optimized for pH, amount of derivatization reagent, time, and temperature. Extraction methods of the acetaldehyde‐hydrazone (AcH‐DNP) stable derivative and product stability studies were carried out. Acetaldehyde was identified by its retention time in comparison with AcH‐DNP standard, using a new chromatography gradient program, and quantitated based on external reference standards and standard addition calibration curves in the presence and absence of ethanol. Results: Derivatization of acetaldehyde was performed at pH 4.0 with an 80‐fold molar excess of DNPH. The reaction was completed in 40 minutes at ambient temperature, and the product was stable for 2 days. A clear separation of AcH‐DNP from DNPH was obtained with a new 11‐minute chromatography program. Acetaldehyde detection was linear up to 80 μM. The recovery of acetaldehyde was >88% in culture media and >78% in plasma. We quantitatively determined the ethanol‐derived acetaldehyde in hepatoma cells, rat blood and plasma with a detection limit around 3 μM. The accuracy of the method was <9% for intraday and <15% for interday measurements, in small volume (70 μl) plasma sampling. Conclusions: An optimized method for the quantitative determination of acetaldehyde in biological systems was developed using derivatization with DNPH, followed by a short RP‐HPLC separation of AcH‐DNP. The method has an extended linear range, is reproducible and applicable to small‐volume sampling of culture media and biological fluids.     

Free radical generation by neutrophils: a potential mechanism of cellular injury in acute alcoholic hepatitis.

Liver membrane vesicles were prepared from operative liver biopsies from six patient volunteers undergoing abdominal surgery for non-hepatic disease. Neutrophils were extracted from their blood. The liver membrane vesicles were exposed to 1 mmol/l acetaldehyde with or without reduction of the resultant adducts formed. The production of superoxide anion by the neutrophils upon exposure to the liver membrane vesicles prepared from the same patient was assessed by measuring the rate of cytochrome c reduction before and after the addition of superoxide dismutase. Preincubation with acetaldehyde significantly increased superoxide production in response to both the reduced (from 35.5 +/- 7.1 nmol O2-/10(8) cells/min to 128 +/- 25, mean +/- SEM, p less than 0.01) and the non-reduced liver cell membranes (from 17.2 +/- 4.3 to 81 +/- 17, p less than 0.01); 1 mmol/l acetaldehyde alone caused no superoxide production. Neutrophil free radical production in response to acetaldehyde altered hepatocyte membranes could be an important mechanism of cellular injury in acute alcoholic hepatitis.     

Role of acetaldehyde in ethanol-induced elevation of the neuroactive steroid 3alpha-hydroxy-5alpha-pregnan-20-one in rats

Background: Systemic ethanol administration increases neuroactive steroid levels that increase ethanol sensitivity. Acetaldehyde is a biologically active compound that may contribute to behavioral and rewarding effects of ethanol. We investigated the role of acetaldehyde in ethanol‐induced elevations of 3α‐hydroxy‐5α‐pregnan‐20‐one (3α,5α‐THP) levels in cerebral cortex. Methods: Male Sprague–Dawley rats were administered ethanol, and plasma acetaldehyde concentrations were measured by gas chromatography to determine relevant concentrations. Rats were then administered acetaldehyde directly, acetaldehyde plus cyanamide to block its degradation, or ethanol in the presence of inhibitors of ethanol metabolism, to determine effects on 3α,5α‐THP levels in cerebral cortex. Results: Ethanol administration (2 g/kg) to rats results in a peak acetaldehyde concentration of 6‐7 μM at 10 minutes that remains stable for the duration of the time points tested. Direct administration of acetaldehyde eliciting this plasma concentration does not increase cerebral cortical 3α,5α‐THP levels, and inhibition of ethanol‐metabolizing enzymes to modify acetaldehyde formation does not alter ethanol‐induced 3α,5α‐THP levels. However, higher doses of acetaldehyde (75 and 100 mg/kg), in the presence of cyanamide to prevent its metabolism, are capable of increasing cortical 3α,5α‐THP levels. Conclusions: Physiological concentrations of acetaldehyde are not responsible for ethanol‐induced increases in 3α,5α‐THP, but a synergistic role for acetaldehyde with ethanol may contribute to increases in 3α,5α‐THP levels and ethanol sensitivity.     

Acetaldehyde induces granulocyte macrophage colony-stimulating factor production in human bronchi through activation of nuclear factor-kappa B.

Acetaldehyde, a metabolite of alcohol and primary mediator of alcohol-induced asthma, causes bronchoconstriction via histamine release from airway mast cells. Acetaldehyde also is found in cigarette smoke and may cause airway inflammation. The purpose of this study was to determine the effect of acetaldehyde on cytokine production and nuclear factor kappa B (NF-kappa B) activation in human bronchial tissues. Human bronchi were prepared from normal parts of lung tissues resected for lung cancer (n = 11). The bronchi were cultured in the presence of 5 x 10(-4) M of acetaldehyde for 24 hours and the concentrations of eotaxin, granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-5, interleukin-8, and regulated on activation, normal T cells expressed and secreted in cultured supernatants were determined by enzyme-linked immunosorbent assay. Tissues also were immunohistochemically stained for NF-kappa Bp65. Acetaldehyde significantly increased GM-CSF production from human bronchi and nuclear translocation of NF-kappa Bp65 in airway epithelium but had no effects on other cytokines. Our findings suggest that acetaldehyde potentially causes airway inflammation via increased GM-CSF production through nuclear translocation of NF-kappa B.     

Role of Acetaldehyde and Acetate in the Development of Ethanol-Induced Cardiac Lipidosis, Studied in Isolated Perfused Rat Hearts

Isolated perfused rat hearts were used to study the effects of ethanol, acetaldehyde, and acetate on the cellular redox state and fatty acid metabolism in the myocardium. Ethanol had negligible effects on the cellular redox state but at high concentrations depressed the contractile activity and thereby secondarily the oxygen consumption. Acetaldehyde in concentrations below 50 microM had negligible effects on the redox state of the mitochondrial free NAD+/NADH couple, as studied by surface fluorometry of flavins and nicotinamide nucleotides. A reduction of NAD+ was observed with concentrations between 50 and 500 microM, while in the range of 0.5-1 mM the effect was biphasic, i.e., an initial reduction was followed by oxidation concomitantly with an increase in heart rate and peak systolic pressure. Acetate in millimolar concentrations caused in the coronary flow. A mitochondrial acetaldehyde dehydrogenase was revealed in the myocardium, having an apparent Km of 1.1 microM for acetaldehyde. Acetaldehyde in 50-microM concentration had no major effects on the uptake, oxidation, or lipid incorporation of oleate in the myocardium. Acetate in concentrations less than 2 mM did not affect the uptake of oleate into the myocardium, but did inhibit is oxidation and enhance its incorporation into tissue lipids in a dose-dependent manner. 2 mM acetate caused a 91% increase in oleate incorporation into tissue lipids over 30 min. The data can be interpreted as showing that acetaldehyde and acetate, the metabolites of ethanol, have metabolic effects on the myocardium, but only those of acetate are significant in concentrations encountered during ethanol oxidation in vivo. It is probable that acetate is involved in the development of ethanol-induced myocardial lipidosis, inhibiting the oxidation of fatty acids, and channelling them into the esterification pathway.     

Genetic variability of enzymes of alcohol metabolism in human beings

Alcohol is eliminated from the body almost entirely by hepatic metabolism, first to acetaldehyde, then to acetate, and finally to carbon dioxide and water. The time course of elimination is best described by Michaelis-Menten kinetics, and rates of elimination following standard doses of ethanol vary among subjects as much as three-fold. Studies comparing rates of elimination in identical and fraternal twins have shown that about half of the variability is attributable to genetic factors. The principal enzymes responsible for ethanol metabolism are alcohol dehydrogenase and aldehyde dehydrogenase. The reaction catalyzed by alcohol dehydrogenase is the rate-limiting step of the pathway. Human livers contain multiple isoenzymes of alcohol dehydrogenase, which are dimeric molecules arising from the association of two subunits encoded by five different structural genes. Genetic polymorphism at two of these gene loci has been described, and all known homo- and heterodimeric forms of the isoenzymes have now been isolated and characterized. Notably, some of them differ quite strikingly in reactivity toward ethanol. Thus a basis for the genetic variability in alcohol metabolic rate can be found in the kinetic properties of the alcohol dehydrogenase isoenzymes. The efficient oxidation of acetaldehyde by hepatic aldehyde dehydrogenase is essential for ethanol oxidation to continue over time, because the equilibrium of the alcohol dehydrogenase reaction favors the conversion of acetaldehyde to ethanol. Acetaldehyde is a very toxic substance the removal of which makes possible the consumption of large quantities of ethanol frequently imbibed by alcoholics. There are also multiple molecular forms of aldehyde dehydrogenase in liver, and the mitochondrial form is the one principally responsible for acetaldehyde oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetaldehyde-Induced Increase in Paracellular Permeability in Caco-2 Cell Monolayer

Evidence indicates that endotoxin-mediated liver injury plays an important role in the pathogenesis of alcoholic liver disease. Elevated plasma endotoxin level in alcoholics is suggested to be caused by enteric bacterial overgrowth and/or increased intestinal permeability to endotoxin. In this study, the effect of ethanol and acetaldehyde on the paracellular permeability was evaluated in Caco-2 cell monolayers. Ethanol was administered into the incubation medium, whereas acetaldehyde was administered by exposing cell monolayers to vapor phase acetaldehyde, or by direct administration of an acetaldehyde generating system (AGS), ethanol + NAD⁺+ alcohol dehydrogenase. Paracellular permeability was assessed by measuring transepithelial electrical resistance (TER), sodium chloride dilution potential, and unidirectional flux of d -[2-³H]mannitol. Administration of ethanol up to 900 mM produced no significant effect on paracellular permeability. Vapor phase acetaldehyde, generated from 5 to 167 mM acetaldehyde solutions in neighboring wells, resulted in a time- and dose-dependent increase in acetaldehyde concentration (99 to 760 μM) in the buffer bathing cell monolayer. Acetaldehyde induced a reduction of TER and dilution potential, and an elevation of mannitol flux in a time and concentration-related manner, without affecting the ability of cells to exclude trypan blue. Removal of acetaldehyde after 1, 2, or 4 hr treatment and subsequent incubation in the absence of acetaldehyde resulted in a time-dependent reversal of TER to baseline values. Administration of AGS also reduced TER and dilution potential, associated with an increase in mannitol flux. This effect of AGS was prevented by 4-methylpyrazole, an alcohol dehydrogenase inhibitor. These results show that acetaldehyde, but not ethanol, reversibly increases the paracellular permeability of Caco-2 cell monolayer.     

Captopril and Lisinopril Decrease Acetaldehyde Effects upon the Prothrombin Time

Captopril, a thiol-containing antihypertensive drug, and lisinopril, an amino-containing antihypertensive drug, will both prolong the prothrombin time (PT) of Level I plasma. Acetaldehyde, a product of ethanol metabolism, also prolongs PT. In a study to examine the interrelationship between hypertension, hemostasis, and alcoholism, an examination of the impact of acetaldehyde on the effects of captopril and lisinopril upon PT was undertaken. It was observed that the pre-mixing of 7.7 × 10⁻³ M captopril with 40.6 mM acetaldehyde for 30 min at R.T. prior to the addition to plasma results in a prolongation of PT which is less than that caused by acetaldehyde alone. Successive additions of captopril and acetaldehyde to plasma also yield a PT which is less than that of acetaldehyde alone. These data suggest that captopril may partially inactivate and detoxify the acetaldehyde effect on hemostasis upon interaction to form a thiohemiacetal. Captopril may prolong PT by the reduction of the S–S bridges in the coagulation factors. Lisinopril behaves similarly to captopril, prolonging PT. Successive additions of lisinopril and acetaldehyde, or pre-mixtures thereof, to plasma result in a lesser prolongation of clotting time relative to acetaldehyde alone. Since primary amines similar to that of lisinopril readily form Schiff bases with acetaldehyde, these data suggest that both captopril and lisinopril may act to detoxify the acetaldehyde effect upon plasma, albeit by different mechanisms.     

Ethanol metabolism in the generation of new antigenic determinants on liver cells.

Antibodies directed against ethanol altered liver cell components have been detected in the serum of nearly 50% of patients with alcoholic liver disease although the pathogenetic mechanisms are unclear. The importance of ethanol metabolism in the generation of new antigenic determinants on liver cells was investigated by in vivo inhibition of alcohol or acetaldehyde dehydrogenase and an induced cytotoxicity assay. There was a significant reduction in cytotoxicity to hepatocytes isolated from rabbits treated with ethanol 1 g/kg when the metabolism of ethanol to acetaldehyde by alcohol dehydrogenase was inhibited. In contrast when the oxidation of acetaldehyde was inhibited by disulfiram cytotoxicity was significantly enhanced. These results show that ethanol metabolism is integral to the expression of the ethanol related determinant and suggest that an impaired ability to metabolism acetaldehyde could lead to the development of immunological reactions to alcohol altered liver membrane antigens.     

Binding of acetaldehyde to a glutathione metabolite: mass spectrometric characterization of an acetaldehyde-cysteinylglycine conjugate

BACKGROUND: Ethanol administration decreases hepatic glutathione levels and increases urinary sulfhydryl excretion. Ethanol-induced liver injury is blunted by the administration of glutathione precursors. Acetaldehyde generated in the metabolism of ethanol binds to a number of amino acid residues in proteins and peptides, but it does not react readily with glutathione. Due to the possible role of acetaldehyde in cysteine and glutathione homeostasis, we investigated the reaction of acetaldehyde to cysteinylglycine, the dipeptide generated in vivo in the hydrolysis of glutathione by gamma-glutamyltransferase. METHODS: A conjugate between acetaldehyde and cysteinylglycine was generated under physiologically relevant conditions, both in vitro and in vivo. It was separated by a new reverse-phase high-performance liquid chromatography method and identified by electrospray ionization/ion trap tandem mass spectrometric analysis. RESULTS: The conjugate with a stoichiometry of 1:1 between cysteinylglycine and acetaldehyde is most rapidly generated in vitro and was identified by mass spectroscopy as 2-methyl-thiazolidine-4-carbonyl-glycine. This thiazolidine derivative is stable in vitro and in biological fluids of rats. The conjugate was present in high concentrations in the bile of rats pretreated with ethanol and an inhibitor of aldehyde dehydrogenase. CONCLUSIONS: The sequestering of cysteinylglycine by acetaldehyde occurs rapidly under physiologic conditions. Long-lived sulfur-containing biomolecules that incorporate acetaldehyde might affect cysteine and glutathione homeostasis and may also play a protective role by reducing circulating acetaldehyde levels. The acetaldehyde conjugate or its metabolic products could potentially serve as markers of ethanol consumption.