Role of Catalase in In Vitro Acetaldehyde Formation by Human Colonic Contents

Ingested ethanol is transported to the colon via blood circulation, and intracolonic ethanol levels are equal to those of the blood ethanol levels. In the large intestine, ethanol is oxidized by colonic bacteria, and this can lead to extraordinarily high acetaldehyde levels that might be responsible, in part, for ethanol-associated carcinogenicity and gastrointestinal symptoms. It is believed that bacterial acetaldehyde formation is mediated via microbial alcohol dehydrogenases (ADHs). However, almost all cytochrome-containing aerobic and facultative anaerobic bacteria possess catalase activity, and catalase can, in the presence of hydrogen peroxide (H₂O₂, use several alcohols (e.g., ethanol) as substrates and convert them to their corresponding aldehydes. In this study we demonstrate acetaldehyde production from ethanol in vitro by colonic contents in a reaction catalyzed by both bacterial ADH and catalase. The amount of acetaldehyde produced by the human colonic contents was proportional to the ethanol concentration, the amount of colonic contents, and the length of incubation time, even in the absence of added nicotinamide adenine dinucleotide or H₂O₂. The catalase inhibitors sodium azide and 3-amino-1,2,4-triazole (3-AT) markedly reduced the amount of acetaldehyde produced from 22 mM ethanol in a concentration dependent manner compared with the control samples (0.1 mM sodium azide to 73% and 10 mM 3-AT to 67% of control). H₂0₂ generating system [β-D(+)-glucose + glucose oxidase] and nicotinamide adenine dinucleotide induced acetaldehyde formation up to 6- and 5-fold, respectively, and together these increased acetaldehyde formation up to 11-fold. The mean supernatant catalase activity was 0.53 ± 0.1 jumol/min/mg protein after the addition of 10 mM H₂0₂, and there was a significant (p < 0.05) correlation between catalase activity and acetaldehyde production after the addition of the hydrogen peroxide generating system. Our results demonstrate that colonic contents possess catalase activity, which probably is of bacterial origin, and indicate that in addition to ADH, part of the acetaldehyde produced in the large intestine during ethanol metabolism can be catalase dependent.     

Alcohol and Aldehyde Dehydrogenases in Human Esophagus: Comparison With the Stomach Enzyme Activities

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) isoenzymes from surgical esophageal and gastric mucosa were compared by agarose isoelectric focusing. Two prominent ADH forms, designated μ1 (equivalent to the recently reported μ-form) and μ2, were expressed in all the 15 esophagus specimens studied, whereas only four of seven examined gastric specimens exhibited a weak to moderately strong μ1-ADH activity band on the isoelectric focusing gels. p l values of the esophageal μ1-ADH and μ2-ADH, and the liver π-ADH were determined to be 8.61, 8.13, and 8.90, respectively. μ-ADHs exhibited high K_m for ethanol (12 mM) and low sensitivity to 4-methylpyrazole inhibition. ALDH3 (BB form) and ALDH1 were the major high- and low-K_m aldehyde dehydrogenase in the esophagus, respectively. The ADH and ALDH activities were determined at pH 7.5 to be 751 ± 78 and 29.9 ± 3.0 nmol/min/g tissue, respectively (measured at 500 mM ethanol or at 200 μM acetaldehyde; mean ± sem ; N = 15). The esophageal ADH activity was approximately 4-fold and the ALDH activity 20% that of the stomach enzyme. Because the presence of high activity and high K_mμ-ADHs as well as low-activity ALDH1 were found in human esophageal mucosa, it is suggested that there may exist an accumulation of intracellular acetaldehyde during alcohol ingestion. This reactive and toxic metabolite may be involved in the pathogenesis of alcohol-induced esophageal disorders.     

Zinc Administration Improves Gastric Alcohol Dehydrogenase Activity and First-Pass Metabolism of Ethanol in Alcohol-Fed Rats

The effects of zinc on first-pass metabolism (FPM) of ethanol and gastric and hepatic alcohol dehydrogenase (ADH) activities have been investigated in two groups of male Wistar rats fed a liquid ethanol diet with normal zinc content (7.6 mg/liter), or zinc supplemented (76 mg/liter), for 21 days, and in two pair-fed groups receiving the same diets without ethanol. Alcoholic rats with normal dietary zinc had lower FPM (1.64 ± 0.25 vs. 2.43 ± 0.20 mM ± hr, p < 0.05) and gastric ADH activity (184 ± 7 vs. 335 ± 41 μmol/min/mg protein, p < 0.01) than control rats. Zinc supplementation did not produce any change in FPM or in gastric ADH activity in control rats. By contrast, in alcoholic rats, the zinc supplement increased gastric ADH activity (247 ± 31 vs. 184 ± 7 μmol/min/mg protein, p < 0.05) and decreased the areas under the curve of blood ethanol concentrations after the intragastric administration of 0.25 g/kg of body weight of ethanol (0.78 ± 0.07 vs. 1.71 ± 0.24 mM ± hr, p < 0.05), thereby increasing the FPM. In conclusion, in alcohol-fed rats, the administration of zinc supplements restores gastric ADH activity and improves the FPM of ethanol. These effects may be one of the mechanisms in which zinc has a beneficial role in preventing the development of alcoholic hepatic lesions.     

In Vitro Alcohol Dehydrogenase-Mediated Acetaldehyde Production by Aerobic Bacteria Representing the Normal Colonic Flora in Man

Excessive ethanol consumption has been related with the development of liver cirrhosis, as well as with rapid intestinal transit time and diarrhea. Moreover, heavy drinking is associated with an increased incidence of cancer of the oropharynx, larynx, esophagus, and colorectum. Acetaldehyde of microbial origin has recently been suggested as a possible pathogenetic factor behind this alcohol-associated gastrointestinal morbidity. The present in vitro study was aimed to investigate alcohol dehydrogenase activity and acetaldehyde formation capacity of some major aerobic bacteria representing the normal colonic flora in man. Cytosolic alcohol dehydrogenase activity and cytosolic protein concentration were determined spectrophotometrically. Alcohol dehydrogenase activity was then calculated as nmoles of reduced substrate produced by milligrams of protein per minute. The ability of different bacteria to produce acetaldehyde was determined by incubating the intact bacterial suspension in closed vials containing ethanol (final concentration 22 mM)for 1 hr at 37°C. The acetaldehyde formed during the incubation was analyzed by headspace gas chromatography. Marked differences in the alcohol dehydrogenase activity and acetaldehyde forming capacity were found among the strains tested. The alcohol dehydrogenase activity varied from 606 ± 91 nmol/min/mg protein ( Escherichia coli IH 50546) to 1 ± 0.2 nmol/min/mg protein ( E. coli IH 50817), and acetaldehyde formation varied from 1,717 ± 2 nmol acetaldehyde/10⁹ colony-forming units ( Klebsiella oxytoca IH 35403) to 5 ± 2 nmol acetaldehyde/10⁹ colony-forming units ( Pseudomonas aeruginosa ATCC 27853). There was a statistically significant correlation ( r = 0.77; p < 0.001) between alcohol dehydrogenase activity and acetaldehyde production from ethanol, strongly suggesting the catalytic role of bacterial alcohol dehydrogenase in this reaction.     

Ethanol Metabolism in Deermice: Role of Extrahepatic Alcohol Dehydrogenase

The relative contributions to ethanol metabolism of extrahepatic alcohol dehydrogenase (ADH) and of liver microsomes were assessed in deermice, which lack hepatic low K _m ADH (ADH^-). In vitro kinetic studies showed the existence of high K _m (>1 M) ADH activity in the liver and kidney, and an enzyme with intermediate K _m in the gastric mucosa ( K _m = 133 mM), whereas the low K _m ADH was missing. With deuterated ethanol, ADH^- deermice showed a significant exchange of reducing equivalents that had been equated with ethanol metabolism by others, whereas we found a poor correlation between the rate of exchange and the rate of metabolism. In vitro studies with subcellular fractions, isolated hepatocytes, and tissue slices revealed that neither liver, nor kidney, nor stomach from ADH^- deermice contributed to exchange of reducing equivalents. These findings clearly indicated that the ADHs with high or intermediate K _m of the tissues studied are not responsible for the exchange. Furthermore, gastrectomized ADH^- deermice still showed an exchange of reducing equivalents, thereby dissociating exchange from gastric ADH activity. Moreover, pretreatment with cimetidine (50 mg/kg body weight), an inhibitor of gastric ADH, did not alter the rate of total ethanol elimination when ethanol was given intraperitoneally. In conclusion, when ethanol is given parenterally, the microsomal ethanol-oxidizing system rather than gastric ADH is a major pathway of ethanol oxidation in ADH^- deermice, whereas both pathways contribute significantly to the metabolism of orally administered ethanol.     

Concentration Dependence of Ethanol Metabolism In Vivo in Rats and Man

The metabolism of ethanol to acetaldehyde has been widely considered to be almost independent of concentration (i.e., "pseudolinear") except when blood ethanol was near the K_m (0.5–1.0 m/ M ) of alcohol dehydrogenase (ADH). On the contrary, we report the concentration dependency of ethanol metabolism, in rats and man, at blood ethanol levels several fold the K_m of ADH. After intravenous loading, blood ethanol disappearance in 10 rats at blood levels between 38 and 17 m/ M ethanol exceeded that between 17 and 4 m/ M (14.09 ± 1.38 versus 8.80 ± 0.86 mmoles/liter blood water/hr ± SEM; p < 0.001). Similarly, in 12 men, blood ethanol disappeared faster at 30–17 m/ M ethanol compared to 17–4 m/ M (5.96 ± 0.33 versus 4.96 7plusmn; 0.28 mmole/liter blood water/hr ± SEM; p < 0.001). When ethanol was maintained at either the 30–60 m/ M or 3–19 m/ M ethanol range in the blood of 14 fasted rats by constant intravenous infusion, ethanol oxidation was 25% greater at the higher concentration (9.08 ± 0.50 versus 7.23 ± 0.41 mmole/liter blood water/hr ± SEM; p < 0.02). Faster ethanol oxidation at high ethanol concentration could be due to the presence of the microsomal ethanol-oxidizing system (MEOS), which would only be fully engaged, because of its K_m (8.5 m/ M ), at ethanol levels exceeding those necessary to saturate ADH. The concentration dependency of ethanol metabolism casts doubt on the validity of the common medicolegal practice of calculating prior blood ethanol levels by linear extrapolation of subsequent ones, on the false assumption that metabolism is unaffected by concentration.     

NMR Investigation of the Futile Cycling of Ethanol in Chronic Alcoholic Rats

Weight gain efficiency differences previously reported between alcohol-fed rats and their controls were investigated. Additionally, the futile cycling of ethanol proposed to explain such differences was studied by NMR spectroscopy. Male Sprague-Dawley rats were fed a nutritionally adequate diet containing 36% of the calories as alcohol, and their paired controls were fed an isocaloric diet for 1 f weeks to establish conditions of chronic alcohol feeding. Normalized metabolic efficiencies varied significantly during the initial 2-week period (6.86 ± 0.51 vs. 2.83 ± 0.18 g/kcal × 10⁻²) for control and alcohol-fed groups, respectively, and to a lesser extent over the entire feeding period (6.41 ± 0.78 vs. 4.60 ± 0.27 g/kcal × 10⁻²) for control and alcohol-fed groups, respectively. Alcohol-induced weight gain inefficiency in metabolism has previously been studied and explained by a variety of different biochemical and physiological mechanisms. One possible pathway of energy wastage may occur due to ethanol futile cycling from ethanol to acetaldehyde through the microsomal ethanol oxidation system pathway, and simultaneously from acetaldehyde to ethanol via the ADH pathway. This futile cycle represents a net loss of 6 ATP/cycle, corresponding to the loss of two reducing equivalents (NADH and NADPH). 1H NMR spectroscopy was used to test for this cycling in blood extracts after administration of 1,1-²H₂ ethanol. No futile cycling was detected either during the initial 2 weeks of feeding or after the entire feeding period.     

Intralobular Distribution of Class I Alcohol Dehydrogenase and Aldehyde Dehydrogenase 2 Activities in the Hamster Liver

The hepatic lobular localization of class I alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) 2 activities was examined histochemically using livers of hamsters with high ethanol preferences. The activity of class I ADH detected by the nitro blue tetrazolium method using 5 mM ethanol as a substrate was extremely high and was almost homogeneously distributed throughout the lobule. The ALDH 2 activity (substrate, 8 μM acetaldehyde) was localized to the centrilobular zone, whereas low K_m ALDH (ALDH 1 + ALDH 2) activity (substrate, 50 μM acetaldehyde) showed a gradient distribution in the lobule with high centrilobular to moderate periportal activity, suggesting that the ALDH 1 activity was distributed throughout the lobule.     

Human Hepatic Alcohol Dehydrogenase and Human Erythrocyte Catalase Do Not Metabolize the Cytochrome P-4502E1 Substrate, Chlorzoxazone

Studies of cytochrome P-4502E1 (CYP2E1)-mediated oxidation of ethanol have been hampered by the lack of a suitable probe for in vivo human studies. Chlorzoxazone, a prescribed skeletal muscle relaxant, is metabolized to 6-hydroxychlorzoxazone by CYP2E1 and has been advocated as a specific probe of this enzyme on the basis of microsomal studies. The applications of this probe may include delineating the contribution of CYP2E1 to in vivo human ethanol metabolism. However, the activity of nonmicrosomal enzymes in metabolizing chlorzoxazone is unknown. Alcohol dehydrogenase (ADH), predominantly a hepatic cytosolic enzyme, may be more important than CYP2E1 in the oxidation of ethanol to acetaldehyde. The contribution of catalase in the in vivo oxidation of ethanol to acetaldehyde is controversial. To determine if either of these enzymes metabolizes chlorzoxazone and whether ethanol oxidation by either enzyme is inhibited by chlorzoxazone or its metabolite, multiple in vitro studies were performed. ADH enzyme kinetics were performed with human recombinant β₁β₁ and β₃β₃ ADH with ethanol and chlorzoxazone (0.5 to 2.5 mM). Neither ADH isoenzyme exhibited NAD⁺-dependent oxidation of chlorzoxazone, but displayed Michaelis-Menten kinetics for ethanol with K_m values of 89 μM and 34 mM, for β₁β₁, and β₃β₃, respectively. Typical in vivo concentrations of chlorzoxazone and its metabolite, 6-hydroxychlorzoxazone, did not alter β₁β₁, or β₃β₃ ADH-mediated oxidation of ethanol to acetaldehyde. Studies of human hepatic nonmicrosomal enzyme activity were expanded to include all nonmicrosomal NAD⁺-dependent hepatic enzymes by starch gel electrophoresis assessment. Human hepatic enzymatic activity in the presence of chlorzoxazone was similar to that observed in the control sample (no added substrate), suggesting a lack of metabolism by NAD⁺-dependent enzymes. Similarly, human erythrocyte catalase, in the presence of a hydrogen peroxide generating system, did not metabolize chlorzoxazone. Furthermore, neither chlorzoxazone nor 6-hydroxychlorzoxazone altered the catalase-induced formation of acetaldehyde from ethanol. These data are consistent with chlorzoxazone as a specific probe of CYP2E1 that may be useful to alcohol researchers.     

Acetaldehyde production and other ADH-related characteristics of aerobic bacteria isolated from hypochlorhydric human stomach

BACKGROUND: Acetaldehyde is a known local carcinogen in the digestive tract in humans. Bacterial overgrowth in the hypochlorhydric stomach enhances production of acetaldehyde from ethanol in vivo after alcohol ingestion. Therefore, microbially produced acetaldehyde may be a potential risk factor for alcohol-related gastric and cardiac cancers. This study was aimed to investigate which bacterial species and/or groups are responsible for acetaldehyde formation in the hypochlorhydric human stomach and to characterize their alcohol dehydrogenase (ADH) enzymes. METHODS: After 7 days of treatment with 30 mg of lansoprazole twice a day, a gastroscopy was performed on eight volunteers to obtain hypochlorhydric gastric juice. Samples were cultured and bacteria were isolated and identified; thereafter, their acetaldehyde production capacity was measured gas chromatographically by incubating intact bacterial suspensions with ethanol at 37 degrees C. Cytosolic ADH activities, Km values, and protein concentration were determined spectrophotometrically. RESULTS: Acetaldehyde production of the isolated bacterial strains (n = 51) varied from less than 1 to 13,690 nmol of acetaldehyde/10(9) colony-forming units/hr. ADH activity of the strains that produced more than 100 nmol of acetaldehyde/10(9) colony-forming units/hr (n = 23) varied from 3.9 to 1253 nmol of nicotinamide adenine dinucleotide per minute per milligram of protein, and Km values for ethanol ranged from 0.65 to 116 mM and from 0.5 to 3.1 M (high Km). There was a statistically significant correlation (r = 0.64, p < 0.001) between ADH activity and acetaldehyde production from ethanol in the tested strains. The most potent acetaldehyde producers were Neisseria and Rothia species and Streptococcus salivarius, whereas nearly all Stomatococcus, Staphylococcus, and other Streptococcus species had a very low capacity to produce acetaldehyde. CONCLUSIONS: This study demonstrated that certain bacterial species or groups that originate from the oral cavity are responsible for the bulk of acetaldehyde production in the hypochlorhydric stomach. These findings provide new information with the respect to the local production of carcinogenic acetaldehyde in the upper digestive tract of achlorhydric human subjects.     

No sex and age influence on the expression pattern and activities of human gastric alcohol and aldehyde dehydrogenases

BACKGROUND: Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are the principal enzymes responsible for ethanol metabolism in humans. The stomach is involved in the metabolism of alcohol during absorption. Conflicting reports exist with regard to the influence of sex and age on the activity of ADH in the human gastric mucosa. The purpose of the present study was to determine the effects of age and sex on the expression pattern and activities of stomach ADH and ALDH. METHODS: A total of 115 endoscopic gastric biopsy specimens were investigated from Han Chinese men (n = 70) and women (n = 45) aged 20-79 years with approximately even distribution among 10-year age intervals. The expression patterns of ADH and ALDH were identified by isoelectric focusing, and the activities were assayed spectrophotometrically. RESULTS: The expression patterns of gastric ADH and ALDH remained unchanged with respect to sex and age. At 33 mM or 500 mM ethanol, pH 7.5, the ADH activities did not differ significantly among the various age groups or between men and women. At 200 microM or 20 mM acetaldehyde, the ALDH activities did not differ significantly in relation to sex and age. No correlations were found between the ADH or ALDH activities at both the high and low substrate concentrations and the ages in men and women. CONCLUSIONS: The results indicate that there is no significant effect of either sex or age on the expression pattern and activity of ADH and ALDH in the human gastric mucosa. The stomach ADH seems unlikely to account for possible variations in the first-pass metabolism of alcohol with regard to sex and age.     

Enzymatic mechanisms of ethanol oxidation in the brain

BACKGROUND: The exact enzymatic mechanisms of ethanol oxidation in the brain are still unclear. The catalase-mediated oxidation of ethanol was demonstrated in rat brain using incubation of brain homogenates with catalase inhibitors. The role of the alcohol dehydrogenase (ADH) or cytochrome P450-dependent system in this process is possible, but has not been confirmed. The objective of the study was to determine the contribution of the different enzymatic pathways to ethanol oxidation in brain homogenates from mice and rats. METHODS: Three approaches were used to investigate the enzymatic mechanisms of ethanol oxidation in the brain of rats and mice: (1) preincubation of brain homogenates with inhibitors of the ethanol-metabolizing enzymes (catalase, CYP2E1, ADH, and ALDH); (2) utilization of mice with genetic deficiency in ethanol-metabolizing enzymes (catalase, CYP2E1, or both enzymes); and (3) determination of ethanol oxidation in brain subcellular fractions known to have differential activity of ethanol-metabolizing enzymes. The ethanol-derived acetaldehyde (AC) and acetate were determined in brain samples by gas chromatography. RESULTS: The catalase inhibitors sodium azide (5 mM) and aminotriazole (5 mM) as well as CYP2E1 inhibitors diallyl sulfide (2 mM) and beta-phenethyl isothiocyanate (0.1 mM) lowered significantly the accumulation of the ethanol-derived AC and acetate in brain homogenates. The ADH inhibitor 4-methyl pyrazole (5 mM) significantly decreased the acetate but not the AC accumulation. Ethanol-derived AC accumulation in brain homogenates of acatalasemic mice was 47% of the control value, 91% in CYP2E1-null mice, and 24% in double mutants (with deficiency of both catalase and CYP2E1). The highest levels of ethanol oxidation were found in microsomal and peroxisomal subcellular brain fractions, where CYP2E1 and catalase are located, respectively. CONCLUSIONS: Catalase is the key enzyme of ethanol oxidation in the brain of rodents: it may be responsible for about 60% of the process. CYP2E1 plays an important role in ethanol oxidation in the rodent brains. Alcohol dehydrogenase plays a minor role, if any, in this process. Aldehyde dehydrogenase plays the crucial role in the further oxidation of ethanol-derived AC in the brain homogenates.     

The Activity of Class I,III, and IV of Alcohol Dehydrogenase Isoenzymes and Aldehyde Dehydrogenase in Gastric Cancer

The metabolism of cancer is in many way different than in healthy cells. Increased metabolism of several carcinogenic substances may take place in cancer cells. The one of them was ethanol, that is oxidized by alcohol dehydrogenase (ADH) to high concentration of acetaldehyde, a toxic and carcinogenic compound. The enzyme responsible for oxidation of acetaldehyde is aldehyde dehydrogenase (ALDH). The aim of this study was to compare the capacity for ethanol metabolism measured by ADH isoenzymes and ALDH activity between gastric cancer and normal gastric mucosa. Total ADH activity was measured by photometric method with p-nitrosodimethylaniline (NDMA) as a substrate and ALDH activity by the fluorometric method with 6-methoxy-2-naphtaldehyde as a substrate. For the measurement of the activity of class I isoenzymes, we used fluorometric methods, with class-specific fluorogenic substrates. The activity of class III ADH was measured by the photometric method with n-octanol and class IV with m-nitrobenzaldehyde as a substrate. The samples were taken surgically during routine operations of gastric carcinomas from 55 patients. The activities of total ADH, and the most important in gastric mucosa, class IV ADH were significantly higher in cancer cells than in healthy tissues. The other tested classes of ADH and ALDH showed a tendency toward higher activity in cancer than in healthy mucosa. The activities of all tested enzymes and isoenzymes were not significantly higher in men than in women in wither gastric cancer tissues or normal mucosa. The increased ADH IV activity may be 1 of the factors intensifying carcinogenesis by the increased ability to acetaldehyde formation from ethanol.     

Characteristics of Alcohol Dehydrogenases of Certain Aerobic Bacteria Representing Human Colonic Flora

We have recently proposed the existence of a bacteriocolonic pathway for ethanol oxidation [i.e., ethanol is oxidzed by alcohol dehydrogenases (ADHs) of intestinal bacteria resulting in high intracoIonic levels of reactive and toxic acetaldehyde]. The aim of this in vitro study was to characterize further ADH activity of some aerobic bacteria, representing the normal human colonic flora. These bacteria were earlier shown to possess high cytosolic ADH activities (Escherichia coli IH 133369, Klebsiella pneumoniae IH 35385, Kleb-siella oxytoca IH 35339, Pseudomonas aeruginosa IH 35342, and Hafnia ahrei IH 53227). ADHs of the tested bacteria strongly preferred NAD as a cofactor. Marked ADH activities were found in all bacteria, even at low ethanol concentrations (1.5 mM) that may occur in the colon due to bacterial fermentation. The K_m for ethanol varied from 29.9 mM for K. pneumoniae to 0.06 mM for Hafnia ahrei. The inhibition of ADH by 4-methylpyrazole was found to be of the competitive type in 4 of 5 bacteria, and K₁ varied from 18.26 ± 3.3 mM for Eschmbhia coli to 0.47 ± 0.13 mM for K. pneumoniae. At pH 7.4, ADH activity was significantly lower than at pH 9.6 in four bacterial strains. ADH of K. oxytoca, however, showed almost equal activities at neutral pH and at 9.6. In conclusion, NAD-linked alcohol dehydrogenases of aerobic colonic bacteria possess low apparent K_m's for ethanol. Accordingly, they may oxidize moderate amounts of ethanol ingested during social drinking with nearly maximal velocity. This may result in the marked production of intracolonic acetaldehyde. Kinetic characteristics of the bacterial enzymes may enable some of them to produce acetaldehyde even from endogenous ethanol formed by other bacteria via alcoholic fermentation. The microbial ADHs were inhibited by 4-methylpyrazole by the same competitive inhibition as hepatic ADH, however, with nearly 1000 times lower susceptibility. Individual variations in human colonic flora may thus contribute to the risk of alcohol-related gastrointestinal morbidity, such as diahea, colon polyps and cancer, and liver injury.     

Helicobacter Infection and Gastric Ethanol Metabolism

The organism frequently colonizing the stomach of patients suffering from chronic active gastritis and peptic ulcer disease– Helicobacter pylori –possesses marked alcohol dehydrogenase (ADH) activity. Consequently, Helicobacter infection may contribute to the capacity of the stomach to metabolize ethanol and lead to increased acetaldehyde production. To study this hypothesis, we first determined ADH activity in a variety of H. pylori strains originally isolated from human gastric mucosal biopsies. ADH activity was also measured in endoscopic gastric mucosal specimens obtained from H. pylori -positive and -negative patients. Furthermore, we used a mouse model of Helicobacter infection to determine whether infected animals exhibit more gastric ethanol metabolism than noninfected controls.
Most of the 32 H. pylori strains studied possessed clear ADH activity and produced acetaldehyde. In humans, gastric ADH activity of corpus mucosa did not differ between H. pylori -positive and -negative subjects, whereas in antral biopsies ADH activity was significantly lower in infected patients. In mice, gastric ADH activity was similar or even lower in infected animals than in controls, depending on the duration of infection, despite the fact that the infectious agent used– Helicobacter felis –showed ADH activity in vitro. In accordance with this, Helicobacter infection tended to decrease rather than increase gastric ethanol metabolism in mice. In humans, it remains to be established whether the observed decrease in antral ADH activity associated with H. pylori infection can lead to reduced gastric first-pass metabolism of ethanol.     

Purification and Partial Amino Acid Sequence of a High-Activity Human Stomach Alcohol Dehydrogenase

To understand the relative importance of alcohol dehydrogenase (ADH) isoenzymes in gastric ethanol metabolism, a stomach-specific ADH (σ-ADH) was purified to homogeneity from human transplant donor and surgical tissues, and its activity for ethanol oxidation was examined. The enzyme from these tissues had a specific activity at pH 10 of ˜70 units/mg, about 10 times that reported by Moreno and Parés ( J. Biol. Chem. 266:1128–1133, 1991). The enzyme exhibited a high K _m for ethanol at pH 7.5 and 10 (29 and 5.2 mM, respectively). This high-activity α-ADH isoenzyme migrated on starch and isoelectric focusing gels to a position slightly anodic to the liver σ isoenzyme. It was subjected to digestion by endoproteinases, and ˜40% of the protein was sequenced. The σ-ADH exhibited 75%, 68%, and 62% sequence identity to the human class I ( β ₁), II (π), and III (χ) isoenzymes, respectively, and 61% identity to the deduced ADH6 amino acid sequence. Phylogenetic analysis indicated that precursors to this high-activity σ-ADH and the class I isoenzymes diverged more recently than precursors to the class II and III isoenzymes, after reptilian and avian divergence. The high-activity σ-ADH isoenzyme therefore represents a distinct class of ADH (class IV), more closely related in evolution to the class I isoenzymes than to the other known human isoenzymes.     

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 10₉ calvaria fibroblasts of newborn rats) and reacted with either 250 mM acetaldehyde and 100 mM CNBH₃ or with 170;μM 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 μM 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.     

Divergence of Ethanol and Acetaldehyde Kinetics and of the Disulfiram-Alcohol Reaction between Subjects with and without Alcoholic Liver Disease

Despite standardization, marked interindividual variation in the severity of the disulfiram-alcohol reaction (DAR) has been observed. We studied the DAR in 51 consecutive alcoholics with ( n = 16) and without ( n = 35) significant alcoholic liver disease. Clinical signs of the DAR were much weaker in the patients with compared with those patients without liver disease. Because acetaldehyde is thought to be the main cause of the DAR, we studied ethanol and acetaldehyde kinetics in 13 patients (6 females, 7 males) with alcoholic liver disease (documented by biopsy, clinical and/or radiological findings, and by quantitative liver function) [galactose elimination capacity (GEC) 4.2 ± SD 1.0 mg/min/kg; aminopyrine breath test (ABT) 0.14 ± 0.10% dose × kg/mmol CO₂] and 13 age- and sex-matched controls (alcoholics without significant liver disease, GEC 7.1 ± 0.7; ABT 0.81 ± 0.35). Clinical signs of acetaldehyde toxicity during the DAR (flush, nausea, tachycardia, and blood pressure drop) were absent in alcoholic liver disease, but clearly evident in controls. Blood ethanol kinetics were similar in both groups, C_max and area under the concentration-time curve (AUC) being 6.27 ± 1.82 and 368.9 ± 72.9 mmol × min/liter in alcoholic liver disease, and 6.62 ± 1.71 and 377.6 ± 124.5 in controls, respectively. In contrast, there was a strong ( p < 0.001) difference in C_max and AUC of acetaldehyde, respective values being 33.46 ± 21.52 and 1463.8 ± 762.5 μmol × min/liter in alcoholic liver disease, and 110.87 ± 56.00 and 4162.0 ± 2424.6 in controls. We hypothesize that the lack of disulfiram-induced acetaldehyde retention in the alcoholic liver disease group may be due to decreased formation of disulfiram metabolites.     

Ethnic Differences in Gastric α-Alcohol Dehydrogenase Activity and Ethanol First-Pass Metabolism

We assessed whether the low α-alcohol dehydrogenase (ADH) activity in Japanese (compared with Caucasians) affects the first-pass metabolism of ethanol. ADH isozyme activities were determined in endoscopic biopsies of the gastric corpus from 24 Japanese and 41 Caucasian men by starch gel electrophoresis and by comparing the reduction of m-nitrobenzaldehyde (a preferred substrate of α-ADH) with that of acetaldehyde (a preferred substrate of γ-ADH) and the glutathione-dependent formaldehyde oxidation (a specific reaction of χ-ADH). Alcohol pharmacokinetics was compared in 10 Japanese and 10 Caucasians after administration of ethanol (300 mg/kg of body weight) intravenously or orally, using 5 and 40% oral solutions. Japanese exhibited lower α-ADH activity than Caucasians, with no difference in the other gastric isozymes. With 5% ethanol, first-pass metabolism was strikingly lower in Japanese than in Caucasions. Blood alcohol levels were similar because of the high elimination rate in Japanese due to the hepatic β₂-ADH variant. With 40% ethanol, the first-pass metabolism increased in both groups to comparable levels, suggesting an additional contribution by χ-ADH at high ethanol concentrations. These results indicate that α-ADH activity contributes significantly to gastric ethanol oxidation and its lower activity in Japanese is associated with lesser first-pass metabolism.     

Functional assessment of human alcohol dehydrogenase family in ethanol metabolism: significance of first-pass metabolism

BACKGROUND: Alcohol dehydrogenase (ADH) is the principal enzyme responsible for ethanol metabolism in mammals. Human ADH constitutes a unique complex enzyme family with no equivalent counterpart in experimental rodents. This study was undertaken to quantitatively assess relative contributions of human ADH isozymes and allozymes to hepatic versus gastric metabolism of ethanol in the context of the entire family. METHODS: Kinetic parameters for ethanol oxidation for recombinant human class I ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, and ADH1C2; class II ADH2; class III ADH3; and class IV ADH4 were determined in 0.1 M sodium phosphate at pH 7.5 over a wide range of substrate concentrations in the presence of 0.5 mM NAD+. The composite numerical formulations for organ steady-state ethanol clearance were established by summing up the kinetic equations of constituent isozymes/allozymes with the assessed contents in livers and gastric mucosae with different genotypes. RESULTS: In ADH1B*1 individuals, ADH1B1 and ADH1C allozymes were found to be the major contributors to hepatic-alcohol clearance; ADH2 made a significant contribution only at high ethanol levels (> 20 mM). ADH1B2 was the major hepatic contributor in ADH1B*2 individuals. ADH1C allozymes were the major contributor at low ethanol (< 2 mM), whereas ADH1B3 the major form at higher levels (> 10 mM) in ADH1B*3 individuals. For gastric mucosal-alcohol clearance, the relative contributions of ADH1C allozymes and ADH4 were converse as ethanol concentration increased. It was assessed that livers with ADH1B*1 may eliminate approximately 95% or more of single-passed ethanol as inflow sinusoidal alcohol reaches approximately 1 mM and that stomachs with different ADH1C genotypes may remove 20% to 30% of single-passed alcohol at the similar level in mucosal cells. CONCLUSIONS: This work provides just a model, but a strong one, for quantitative assessments of ethanol metabolism in the human liver and stomach. The results indicate that the hepatic-alcohol clearance of ADH1B*2 individuals is higher than that of the ADH1B*1 and those of the ADH1B*3 versus the ADH1B*1 vary depending on sinusoidal ethanol levels. The maximal capacity for potential alcohol first-pass metabolism in the liver is greater than in the stomach.