Formation Of Acetaldehyde Adducts Research Articles

Evaluation of main post-translational modifications occurring in naturally generated peptides during the ripening of Spanish dry-cured ham.

Peptidyl post-translational modifications (PTMs) could influence the final quality of processed meat. In this study, the peptide oxidative phenomena in Spanish dry-cured ham (Biceps femoris muscle) was evaluated at different ripening times (9, 12, 15, 18 and 24months of processing) evidencing interactions amongst the lipid and protein oxidation, major peptidyl PTMs and the release of free amino acids (FAAs). Results showed that 12months of processing enabled the most abundant protein-bound carbonyls, while TBARS value was significantly favored (p<0.001) by ripening. However, FAAs were still intensively generated during overall ripening. Peptidomics and chemometrics further revealed that proteolysis mostly hampered the oxidized peptides rather than the deamidated ones during ripening. Myosin light chain (MYL1 and MYL3) showed high oxidative susceptibility owing to peptidyl methionine and proline oxidation as well as acetaldehyde adduct formation on lysine or histidine residues.

Relevance of the antioxidant properties of methotrexate and doxycycline to their treatment of cardiovascular disease.

Many medications exhibit clinical benefits that are unrelated to their primary therapeutic uses. In many cases, the mechanisms underpinning these pleotropic effects are unknown. Two commonly prescribed medications that exhibit pleotropic benefits in cardiovascular disease and other diseases associated with chronic inflammation are methotrexate (MTX) and doxycycline (DOX). The vast majority of cardiovascular disease is associated with atherosclerosis. Because atherosclerosis is a chronic inflammatory disease, possible mechanisms by which MTX and DOX reduce inflammation have been investigated. Interestingly, the primary structure of both of these medications contain aromatic phenolic rings, which resemble polyphenols that are known to possess antioxidant activity. Inflammation and oxidative stress are intimately related. Inflammation promotes oxidative stress, which in turn leads to further inflammation; in this way, oxidative stress and inflammation can establish a self-perpetuating cycle. It has been shown that MTX and DOX act as antioxidants and are capable of scavenging free radicals and the reactive oxygen species (ROS) superoxide (O2-). Furthermore, both MTX and DOX inhibit the formation of malondialdehyde acetaldehyde (MAA) adducts, products of oxidative stress and lipid peroxidation. Importantly, MAA-adducts are highly immunogenic and initiate inflammatory responses; thereby, fueling the cycle of inflammation and oxidative stress that results in chronic inflammation. Thus, reducing the formation of MAA-adducts may ameliorate inflammation that leads to ROS production and in this way, break the self-sustaining cycle of oxidative stress and inflammation. It is possible that the under-recognized antioxidant properties of these medications may be a mechanism by which they and other medications provide pleotropic benefit in the treatment of chronic inflammatory disease.

Alcohol metabolism by oral streptococci and interaction with human papillomavirus leads to malignant transformation of oral keratinocytes.

Poor oral hygiene, ethanol consumption, and human papillomavirus (HPV) are associated with oral and esophageal cancers. However, the mechanism is not fully known. This study examines alcohol metabolism in Streptococcus and its interaction with HPV-16 in the malignant transformation of oral keratinocytes. The acetaldehyde-producing strain Streptococcus gordonii V2016 was analyzed for adh genes and activities of alcohol and aldehyde dehydrogenases. Streptococcus attachment to immortalized HPV-16 infected human oral keratinocytes, HOK (HPV/HOK-16B), human oral buccal keratinocytes, and foreskin keratinocytes was studied. Acetaldehyde, malondialdehyde, DNA damage, and abnormal proliferation among keratinocytes were also quantified. We found that S. gordonii V2016 expressed three primary alcohol dehydrogenases, AdhA, AdhB, and AdhE, which all oxidize ethanol to acetaldehyde, but their preferred substrates were 1-propanol, 1-butanol, and ethanol, respectively. S. gordonii V2016 did not show a detectable aldehyde dehydrogenase. AdhE is the major alcohol dehydrogenase in S. gordonii. Acetaldehyde and malondialdehyde production from permissible Streptococcus species significantly increased the bacterial attachment to keratinocytes, which was associated with an enhanced expression of furin to facilitate HPV infection and several malignant phenotypes including acetaldehyde adduct formation, abnormal proliferation, and enhanced migration through integrin-coated basement membrane by HPV-infected oral keratinocytes. Therefore, expression of multiple alcohol dehydrogenases with no functional aldehyde dehydrogenase contributes to excessive production of acetaldehyde from ethanol by oral streptococci. Oral Streptococcus species and HPV may cooperate to transform oral keratinocytes after ethanol exposure. These results suggest a significant clinical interaction, but further validation is warranted.

Inhibition of CYP2E1 leads to decreased malondialdehyde–acetaldehyde adduct formation in VL-17A cells under chronic alcohol exposure

AimEthanol metabolism leads to the formation of acetaldehyde and malondialdehyde. Acetaldehyde and malondialdehyde can together form malondialdehyde–acetaldehyde (MAA) adducts. The role of alcohol dehydrogenase (ADH) and cytochrome P4502E1 (CYP2E1) in the formation of MAA-adducts in liver cells has been investigated. Main methodsChronic ethanol treated VL-17A cells over-expressing ADH and CYP2E1 were pretreated with the specific CYP2E1 inhibitor — diallyl sulfide or ADH inhibitor — pyrazole or ADH and CYP2E1 inhibitor — 4-methyl pyrazole. Malondialdehyde, acetaldehyde or MAA-adduct formation was measured along with assays for viability, oxidative stress and apoptosis. Key findingsInhibition of CYP2E1 with 10μM diallyl sulfide or ADH with 2mM pyrazole or ADH and CYP2E1 with 5mM 4-methyl pyrazole led to decreased oxidative stress and toxicity in chronic ethanol (100mM) treated VL-17A cells. In vitro incubation of VL-17A cell lysates with acetaldehyde and malondialdehyde generated through ethanol led to increased acetaldehyde (AA)-, malondialdehyde (MDA)-, and MAA-adduct formation. Specific inhibition of CYP2E1 or ADH and the combined inhibition of ADH and CYP2E1 greatly decreased the formation of the protein aldehyde adducts. Specific inhibition of CYP2E1 led to the greatest decrease in oxidative stress, toxicity and protein aldehyde adduct formation, implicating that CYP2E1 accelerates the formation of protein aldehyde adducts which can be an important mechanism for alcohol mediated liver injury. SignificanceCYP2E1-mediated metabolism of ethanol leads to increased AA-, MDA-, and MAA-adduct formation in liver cells which may aggravate liver injury.

In Vitro Evidence for Chronic Alcohol and High Glucose Mediated Increased Oxidative Stress and Hepatotoxicity

Hyperglycemia or alcoholism can lead to impaired liver functions. Cytochrome P450 2E1 (CYP2E1) is elevated in hyperglycemia or alcoholism and plays a critical role in generating oxidative stress in the cell. In the present study, we have used VL-17A cells that overexpress the alcohol metabolizing enzymes [alcohol dehydrogenase (ADH) and CYP2E1] to investigate the toxicity due to ethanol (EtOH) plus high glucose. Toxicity was assessed through viability assay and amount of acetaldehyde adduct formation. Oxidative stress parameters included measuring reactive oxygen species (ROS) levels and malondialdehyde adduct formation. Apoptosis was determined through caspase-3 activity, Annexin V- Propidium iodide staining, and changes in mitochondrial membrane potential. The effects of antioxidants and specific inhibitors of ADH and CYP2E1 on cell viability and ROS levels were also studied. When present together, EtOH plus high glucose-treated VL-17A cells exhibited greater oxidative stress and toxicity than other groups. Apoptosis was observed in liver cells treated with the toxins, and the EtOH plus high glucose-treated VL-17A cells exhibited apoptosis to the largest extent. A distinct and graded increase in CYP2E1 level occurred in the different groups of VL-17A cells. Further, antioxidants or inhibitors of ADH and CYP2E1 were effective in decreasing the observed oxidative stress and toxicity. The combined oxidative insult due to alcohol plus high glucose leads to greater liver injury, which may prove to be a timely warning for the injurious effects of alcohol consumption in diabetics.

Acetaldehyde-derived modifications on cytosolic human carbonic anhydrases

Acetaldehyde can generate modifications in several proteins, such as carbonic anhydrase (CA) II. In this study, we extended in vitro investigations on acetaldehyde adduct formation by focusing on the other human cytosolic CA enzymes I, III, VII, and XIII. High-resolution mass spectrometric analysis indicated that acetaldehyde most efficiently formed covalent adducts with CA II and XIII. The binding of up to 19 acetaldehydes in CA II is probably attributable to the high number of lysine residues (n = 24) located mainly on the surface of the enzyme molecule. CA XIII formed more adducts (up to 25) than it contains lysine residues (n = 16) in its primary structure. Acetaldehyde treatment induced only minor changes in CA catalytic activity in most cases. The present study provides the first evidence that acetaldehyde can bind to several cytosolic CA isozymes. The functional consequences of such modifications will be further investigated in vivo by using animal models.

Alcohol and the myocardium.

Structural and functional abnormalities are prominent in alcoholic cardiomyopathy (ACM). Histological features in affected subjects are almost identical to the characteristics of dilated cardiomyopathy. Quantitative morphometry, however, can distinguish between ACM and dilated cardiomyopathy. Biopsies from patients with ACM show increases in the activities of some myocardial enzymes (alpha-hydroxybutyric dehydrogenase, creatine kinase, lactate dehydrogenase, malic dehydrogenase) which are correlated with the bimodal distribution of alcohol intake and may represent an adaptive response. One-third of patients with ACM have serum antibodies against cardiac acetaldehyde-protein adducts. Animal models of ethanol toxicity have shown that acutely, alcohol and acetaldehyde reduce the synthesis of cardiac contractile proteins in vivo. Two-dimensional SDS-PAGE has also shown that in rats chronically fed alcohol, the relative amounts of over 10% of heart muscle proteins are altered. The heat shock proteins (HSP) Hsp60 and Hsp70 are decreased in alcohol-fed rats, as is desmin. Reduction in HSPs may indicate reduced myocardial protection whilst a fall in desmin may indicate structural defects. In conclusion, ACM is a complex process that is due to altered protein synthesis, the formation of acetaldehyde adducts and a reduction of cardiac HSPs and desmin. Both acetaldehyde and alcohol are myocardial perturbants.

Sequence Distribution of Acetaldehyde-Derived N2-Ethyl-dG Adducts along Duplex DNA

Acetaldehyde (AA) is the major metabolite of ethanol and may be responsible for an increased gastrointestinal cancer risk associated with alcohol beverage consumption. Furthermore, AA is one of the most abundant carcinogens in tobacco smoke and induces tumors of the respiratory tract in laboratory animals. AA binding to DNA induces Schiff base adducts at the exocyclic amino group of dG, N2-ethylidene-dG, which are reversible on the nucleoside level but can be stabilized by reduction to N2-ethyl-dG. Mutagenesis studies in the HPRT reporter gene and in the p53 tumor suppressor gene have revealed the ability of AA to induce G-->A transitions and A-->T transversions, as well as frameshift and splice mutations. AA-induced point mutations are most prominent at 5'-AGG-3' trinucleotides, possibly a result of sequence specific adduct formation, mispairing, and/or repair. However, DNA sequence preferences for the formation of acetaldehyde adducts have not been previously examined. In the present work, we employed a stable isotope labeling-HPLC-ESI+-MS/MS approach developed in our laboratory to analyze the distribution of acetaldehyde-derived N2-ethyl-dG adducts along double-stranded oligodeoxynucleotides representing two prominent lung cancer mutational "hotspots" and their surrounding DNA sequences. 1,7,NH 2-(15)N-2-(13)C-dG was placed at defined positions within DNA duplexes derived from the K-ras protooncogene and the p53 tumor suppressor gene, followed by AA treatment and NaBH 3CN reduction to convert N2-ethylidene-dG to N2-ethyl-dG. Capillary HPLC-ESI+-MS/MS was used to quantify N2-ethyl-dG adducts originating from the isotopically labeled and unlabeled guanine nucleobases and to map adduct formation along DNA duplexes. We found that the formation of N2-ethyl-dG adducts was only weakly affected by the local sequence context and was slightly increased in the presence of 5-methylcytosine within CG dinucleotides. These results are in contrast with sequence-selective formation of other tobacco carcinogen-DNA adducts along K-ras- and p53-derived duplexes and the preferential modification of endogenously methylated CG dinucleotides by benzo[a]pyrene diol epoxide and acrolein.

Formation of acetaldehyde adducts of glutathione S-transferase A3 in the liver of rats administered alcohol chronically

Hepatic tissue damage induced by chronic exposure to alcohol is mediated through acetaldehyde and associated with reactive oxygen species, which impair cellular defense mechanisms. Because glutathione S-transferases (GSTs) play an important role in the detoxification of xenobiotics and reactive oxygen species, the current study was undertaken to test the effect of alcohol administration on structural and functional characteristics of rat (r) liver Alpha class rGSTs. Western blot analysis revealed an appreciable change in the expression of rGSTA3 subunit levels, whereas no change was observed in activity after chronic alcohol treatment. Reverse-phase high performance liquid chromatographic analysis of rat liver GSTs that were affinity purified with glutathione showed a 1.07-fold increase in rGSTA3 subunit levels in rats treated with alcohol chronically. In addition, liquid chromatographic–electrospray ionization mass spectrometric analysis of GSTs that were affinity purified with glutathione showed the formation of acetaldehyde adducts to the rGSTA3 subunit. Given the abundant expression of rGSTA3 subunit and acetaldehyde adduct formation, results of the current study support the suggestion that modification of rGSTA3 subunit, and thus its impaired function, in alcohol-exposed rats may contribute to the progression of alcohol-induced liver damage.

Changes in Serum Apolipoprotein and Lipoprotein Profile After Alcohol Withdrawal: Effect of Apolipoprotein E Polymorphism

Apolipoprotein (Apo) E genotype and alcohol consumption or withdrawal strongly affect lipoprotein (Lp) metabolism and, as with any genetic and environmental factors, they might interact. The aim of this study was to investigate this gene/environment interaction by analyzing the effect of the apoE genotype on the alcohol withdrawal-induced alterations in the serum Apo and Lp profile. ApoE genotypes and concentrations of serum cholesterol, triglyceride, and Lps containing apoA-I, A-II, B, E, and C-III were determined in 84 male alcohol abusers before and after 3 weeks of abstinence. After withdrawal, concentrations of serum apoA-I, LpA-I, LpA-I/A-II, apoC-III, LpC-III-non-B, apoE, and LpE-non-B significantly decreased, whereas those of triglycerides and apoB increased; levels of cholesterol, LpC-III:B, and LpB:E were not affected. ANOVA shows that apoE polymorphism effects were quite similar before and after alcohol withdrawal on all serum Apos and Lps (the interaction term between withdrawal and apoE genotype was not significant). The only interaction term that was borderline significant (p < or = 0.10) concerned the apoB concentration. Before withdrawal, no association between apoB level and apoE polymorphism was observed, whereas after abstinence, a borderline significant (p < or = 0.10) gradient of concentration across the three groups of subjects (epsilon2 carriers < epsilon3/epsilon3 < epsilon4 carriers) was noticed. Alcohol abstinence causes major changes in the antiatherogenic Apos and Lps and may increase those known to be atherogenic. Heavy alcohol consumption seems to alter the effect of apoE polymorphism on apoB levels, and further investigations are needed to clarify the mechanisms involved in this phenomenon: a defect in sialylation of apoE, formation of acetaldehyde adducts on apoB, or both.

Malondialdehyde–acetaldehyde-adducted bovine serum albumin activates protein kinase C and stimulates interleukin-8 release in bovine bronchial epithelial cells

Previous study results have demonstrated that cigarette smoke or acetaldehyde rapidly stimulates protein kinase C (PKC)–mediated release of interleukin-8 (IL-8) in bovine bronchial epithelial cells (BECs). Low concentrations of acetaldehyde combine synergistically with malondialdehyde to increase significantly maximal BEC PKC activity at 48 to 96 h stimulation. Because more than 95% of alcoholics are cigarette smokers, we hypothesized that malondialdehyde, an inflammation product of lipid peroxidation, and acetaldehyde, both a product of ethanol metabolism and a component of cigarette smoke, might stimulate PKC-mediated IL-8 release in BECs by malondialdehyde–acetaldehyde (MAA) adduct formation, rather than as free aldehydes. Protein kinase C activity is maximally elevated in BECs treated with 50 μg/ml of BSA–MAA from approximately 1 to 3 h. This activity subsequently begins to decrease by 4 to 6 h, with a return to baseline unstimulated kinase activity levels by 24 h. No activation of cyclic AMP–dependent protein kinase (PKA) or cyclic GMP–dependent protein kinase (PKG) was observed in BSA–MAA-treated BECs. The MAA adduct activation of PKC was followed by a fourfold to tenfold greater release of IL-8 over that observed for both BECs exposed to media only and BSA control–treated BECs. Protein kinase C activation and IL-8 release were blocked by pretreating BECs with 1 μM calphostin C or 100 nM of the PKC alpha–specific inhibitor, Gö 6976. Isoform-specific inhibitors to PKC beta, PKC delta, and PKC zeta failed to inhibit completely MAA adduct–stimulated PKC or IL-8 release. Results of these studies indicate that metabolites derived from ethanol and cigarette smoke, such as acetaldehyde and malondialdehyde, form adducts that stimulate airway epithelial cell PKC alpha–mediated release of promigratory cytokines.

Experimental heart muscle damage in alcohol feeding is associated with increased amounts of reduced‐ and unreduced‐acetaldehyde and malondialdehyde–acetaldehyde protein adducts

Chronic and excessive alcohol consumption induces defined myocardial lesions characterized by impaired structural, mechanical and biochemical features. The pathogenic mechanisms are unknown, although it is possible that protein adduct formation by reactive metabolites of ethanol may be a contributory process. Hitherto, this has only been tested with respect to antibodies against reduced-acetaldehyde protein adducts in clinical studies, despite the fact that during alcohol toxicity the formation of reduced-acetaldehyde, unreduced-acetaldehyde, malondialdehyde, malondialdehyde-acetaldehyde and hydroxyethyl protein adducts have been reported in non-cardiac tissues. It was our hypothesis that the heart is particularly sensitive to the formation of protein adducts in alcohol toxicity.To test this hypothesis, we analysed hearts from rats fed nutritionally complete liquid diets containing ethanol as 35% of total calories for 6 weeks, using the Lieber-DeCarli pair-feeding protocol. Control rats were treated identically and fed the same diet in which ethanol was replaced by isocaloric glucose. At the end of the feeding period, the hearts were dissected and ventricular muscle analysed. After 6 weeks' ethanol feeding, ELISA analysis showed increased amounts of reduced-acetaldehyde protein adducts (p < 0.01) unreduced-acetaldehyde (p < 0.01) and malondialdehyde-acetaldehyde (p= 0.01) protein adducts. However, malondialdehyde and alpha-hydroxyethyl-protein adducts were not significantly increased in hearts of ethanol-fed rats compared to pair-fed control (p > 0.1 in both instances). This is the first report of acetaldehyde adduct formation in alcoholic cardiomyopathy. This suggests that either immune process may develop or functional impairment of affected proteins may occur.

MECHANISMS OF HEPATIC INJURY IN ALCOHOLIC LIVER DISEASE

Essentially all patients with significant alcoholic liver disease (ALD) have clinical manifestations of malnutrition. 101 Work by Lieber and others, however, has demonstrated that ALD can occur despite adequate nutrition. 85,86 This suggests that the liver injury of ALD is attributable to direct toxic effects of alcohol and its metabolites or the consequences of alcohol metabolism. Major research efforts into these areas have uncovered several potential mechanisms that may play a role in the liver injury of ALD. In all likelihood, several mechanisms operate simultaneously to initiate, potentiate, or perpetuate liver injury once it is initiated in clinical ALD. In addition, the initiating events or factors in alcoholic liver injury and the reasons why some individuals are predisposed to ALD are not known. Although interesting data recently were reported on the mechanisms of liver fibrosis in ALD, alcoholic hepatitis (AH) and acute liver injury are the foci of our discussion because most investigators agree that AH is an intermediate but potentially reversible stage of ALD. It is hoped that further knowledge concerning the pathogenesis of AH will allow the development of more effective treatments for this potentially lethal disease. The concepts of liver injury caused by alcohol-related oxidative stress (lipid peroxidation), immune response (cytokines), fatty acid metabolites, acetaldehyde production (protein–acetaldehyde adduct formation), mitochondrial dysfunction, and synergism of viral liver injuries, have been proposed. 81,145 These are the areas that have received major investigative attention over the past decade and are reviewed in this article.

Collagen Synthesis by Liver Stellate Cells Is Released from Its Normal Feedback Regulation by Acetaldehyde‐Induced Modification of the Carboxyl‐Terminal Propeptide of Procollagen

Acetaldehyde stimulates collagen synthesis in stellate cells and forms adducts with procollagen in the liver of alcoholics. To assess the possibility that modification of the carboxyl-terminal propeptide by acetaldehyde affects its capacity to exert a feedback inhibition of collagen synthesis after splitting from procollagen, the propeptide was prepared by gel filtration of the bacterial collagenase digests of procollagen type I (obtained from 10(9) calvaria fibroblasts of newborn rats) and reacted with either 250 mM acetaldehyde and 100 mM CNBH3 or with 170 microM acetaldehyde without reducing agents, to mimick in vivo conditions. The unmodified propeptide produced a concentration-dependent inhibition of collagen synthesis by Ito cells. By contrast, the acetaldehyde-modified propeptide produced a lesser inhibition of procollagen synthesis in the cells, associated with a greater accumulation of collagen in the media. The incubation with 170 microM acetaldehyde and, to a lesser extent, 50 mM ethanol produced collagenase-digestible adducts in stellate cells. Thus, the formation of acetaldehyde adducts with the carboxyl-terminal propeptide of procollagen may account, at least in part, for the stimulatory effect of acetaldehyde on collagen synthesis by stellate cells and may lead to collagen accumulation through a decrease of the normal feedback regulation of collagen synthesis by the propeptide.

Binding of acetaldehyde to rat gastric mucosa during ethanol oxidation

Acetaldehyde, the first product of ethanol metabolism, has previously been shown to form potentially harmful adducts with various proteins. The aim of this study was to investigate whether acetaldehyde—either exogenous or metabolically derived—binds to gastric mucosal proteins. Homogenized rat gastric mucosa was incubated with various concentrations of radiolabeled acetaldehyde or ethanol for different time periods. Acetaldehyde-protein adducts were determined by a liquid scintillation counter. In addition, mucosa was incubated with nonlabeled ethanol, and the acetaldehyde formed was measured by using headspace gas chromatography. Incubation of gastric mucosa with ( 14C)-acetaldehyde led to a concentration- and time- dependent radiolabeling of mucosal proteins. Formation of acetaldehyde adducts occurred relatively rapidly within 30 minutes and even at low acetaldehyde levels (5 μmol/L). Stable adducts represented 77% ± 5% (mean ± SEM) of the total adducts formed. In the presence of ethanol, acetaldehyde production and adduct formation took place in a concentration- and time-dependent manner. 4-Methylpyrazole and sodium azide inhibited acetaldehyde production to 7% ± 1% of control and decreased the amount of acetaldehyde adducts to 55% ± 8%. Enhanced acetaldehyde formation (to 420% ± 50%) was clearly reflected in increased adduct formation (550% ± 110%). In conclusion, both exogenous and endogenous acetaldehyde binds to gastric mucosal proteins in vitro. Gastric mucosal acetaldehyde production and the consequent adduct formation could be a pathogenetic factor behind ethanol-associated gastric injury.

Hepatic fibrosis produced in guinea pigs by chronic ethanol administration and immunization with acetaldehyde adducts

Experimental hepatic fibrosis was produced in the guinea pig. We produced hepatic necrosis associated with inflammatory cell infiltration in guinea pigs immunized with acetaldehyde adducts and fed ethanol for 40 days. Extending the period of these treatments to 90 days resulted in producing hepatic fibrosis developing around individual hepatocytes in the terminal hepatic venule areas and portal areas, accompanied by an increase in hepatic hydroxyproline content. In contrast, no fibrosis was observed in the livers of the control groups that had been exposed to nothing, ethanol alone, or a combination of ethanol and immunization with unmodified human hemoglobin. Minimal fibrotic changes were observed in animals immunized with human hemoglobin acetaldehyde adducts but not fed ethanol. These results indicate that the formation of acetaldehyde adducts and the acquisition of immunity against them can produce hepatic fibrosis. Immune mechanisms against acetaldehyde adducts may, in part, be involved in the pathogenesis of hepatic fibrosis seen in alcoholics.

Hepatic fibrosis produced in guinea pigs by chronic ethanol administration and immunization with acetaldehyde adducts

Experimental hepatic fibrosis was produced in the guinea pig. We produced hepatic necrosis associated with inflammatory cell infiltration in guinea pigs immunized with acetaldehyde adducts and fed ethanol for 40 days. Extending the period of these treatments to 90 days resulted in producing hepatic fibrosis developing around individual hepatocytes in the terminal hepatic venule areas and portal areas, accompanied by an increase in hepatic hydroxyproline content. In contrast, no fibrosis was observed in the livers of the control groups that had been exposed to nothing, ethanol alone, or a combination of ethanol and immunization with unmodified human hemoglobin. Minimal fibrotic changes were observed in animals immunized with human hemoglobin acetaldehyde adducts but not fed ethanol. These results indicate that the formation of acetaldehyde adducts and the acquisition of immunity against them can produce hepatic fibrosis. Immune mechanisms against acetaldehyde adducts may, in part, be involved in the pathogenesis of hepatic fibrosis seen in alcoholics.

Separation of Hemoglobin Acetaldehyde Adducts by High-Performance Liquid Chromatography Cation-Exchange Chromatography

A HPLC-based method was developed to provide a simple way to study changes to hemoglobin induced by acetaldehyde in vitro. This method distinguished 18 human hemoglobin fractions including a new acetaldehyde-induced fraction HbA1ach3. The method consists of a Poly CAT A cation-exchange column and a stepwise salt and pH gradient, with a total analysis time of 31 min. The formation of acetaldehyde adducts was studied by incubation of hemoglobin with different Ach concentrations (5-1000 μM) and different incubation times (0-48 h). Physiological (5-250 μM) Ach concentrations induced increases mainly in 3 known fractions: HbA1ach1, HbA1prec, and HbA1d3; plus, it caused the formation of a new fraction, HbA1ach3. The specificity of the changes to acetaldehyde was studied by incubation of hemoglobin with glucose and acetylsalicylic acid. HbA1ach3 was the only acetaldehyde-induced hemoglobin fraction which was not also increased by glucose and acetylsalicylic acid treatment. The formation of HbA1ach3 showed a dose and time dependence on acetaldehyde incubations. Dialyzation and reduction experiments showed that HbA1ach3 is a stable adduct of hemoglobin, and incubation with purified HbA0 showed that HbA1ach3 is an adduct of HbA0. The within-run and between-run coefficients of variation for HbA1ach3 (0.83% of total hemoglobin) were 10.8 and 15.1%, respectively, and the analytical recovery was 82-97%. These results indicate that in addition to the new, acetaldehyde-specific fraction HbA1ach3, several other types of hemoglobin adducts were formed with acetaldehyde. The current method might be useful in clarifying the relationships between hemoglobin and acetetaldehyde in vitro. Moreover, the measurement of HbA1ach3 may form the basis for an improved method for the clinical monitoring of alcohol abusers.

Formation of acetaldehyde adducts and their significance in the pathogenesis of alcoholic liver disease

Formation of acetaldehyde adducts and their significance in the pathogenesis of alcoholic liver disease

THE DELETERIOUS EFFECTS OF ALCOHOL ON THE HEART: INVOLVEMENT OF PROTEIN TURNOVER

Ethanol ingestion can induce a variety of metabolic changes in the heart, and recent studies have even suggested that moderate ethanol intake confers cardio-protective effects in reducing mortality due to coronary artery disease. However, in this review we explore the consequences of excessive ethanol intake or alcohol misuse. We investigate the epidemiology of alcohol misuse, and thereafter present evidence supporting the existence of a specific alcoholic heart muscle disease (AHMD). The prevalence of AHMD is described and the contributing mechanisms are discussed. These include the involvement of free radicals, defects in intermediary metabolism and the formation of acetaldehyde adducts. Special attention has been paid to ethanol-induced changes in protein turnover. Our studies show that ethanol impairs the protein synthetic pathways, affecting the contractile proteins per se, as well as the subcellular organelles as exemplified by the mitochondria. Acetaldehyde appears to be a particularly potent toxin. The experiments described in this review emphasize the need for investigative studies in intact models. The mechanisms inherent in the pathogenesis of AHMD may be applicable to other heart muscle disorders.