Michaelis-Menten Elimination Kinetics of Acetate During Ethanol Oxidation

Background: The redistribution of acetate cannot be explained using a linear kinetic model. We studied the pharmacokinetics of acetate during ethanol oxidation in the rabbit. Methods: An ethanol saline solution (0.5, 1.5, and 2.5 g/kg) was injected bolus intravenously. We measured blood ethanol, acetaldehyde, and acetate concentrations by using head-space gas chromatography. Results: Blood acetate concentration changed in three phases: an ascending, a plateau, and a declining phase. The first-order rate constant of the declining phase was smaller than that of the ascending phase and decreased dose dependently. Statistical moment analysis of the blood acetate profiles showed that the normalized area under the curve (AUC/Dose) and the mean residence time (MRT) increased with increasing dose amount. These increases suggest a capacity-limited elimination of acetate. We attempted simultaneous multiline fitting, using the three blood acetate disappearance curves, to determine the pharmacokinetic model. Consequently, the blood acetate profile was best described by a Michaelis-Menten elimination kinetic model. The V_max and K_m values of acetate elimination were 40.80 ± 14.10 mM/hr and 0.47 ± 0.19 mM, respectively. The fraction of a dose of ethanol converted to acetate (f_AcA) was calculated to be 0.54. The estimated values are average parameter values of three different doses. Fitted curves suggest smaller f_AcA at a low dose and larger f_AcA at a higher dose, which indicate increases of accumulation and redistribution of acetate at higher doses. Conclusions: Acetate elimination during ethanol oxidation obeys capacity-limited kinetics.     

Gender differences in pharmacokinetics of alcohol

BACKGROUND: The enhanced vulnerability of women to develop alcohol-related diseases may be due to their higher blood alcohol levels after drinking, but the mechanism for this effect is debated. METHODS: Sixty-five healthy volunteers of both genders drank 0.3 g of ethanol/kg of body weight (as 5%, 10%, or 40% solutions) postprandially. Blood alcohol concentrations were monitored by breath analysis and compared with those after intravenous infusion of the same dose. First-pass metabolism was quantified (using Michaelis-Menten kinetics) as the route-dependent difference in the amount of ethanol reaching the systemic blood. Gastric emptying was assessed by nuclear scanning after intake of 300 microCurie of technetium-labeled diethylene triamine pentacetic acid in 10% ethanol. The activities of alcohol dehydrogenase isozymes were assessed in 58 gastric biopsies, using preferred substrates for gamma-ADH (acetaldehyde) and for final sigma-ADH (m-nitrobenzaldehyde) and a specific reaction of chi-ADH (glutathione-dependent formaldehyde dehydrogenase). RESULTS: Women had less first-pass metabolism than men when given 10% or 40%, but not 5%, alcohol. This was associated with lower gastric chi-ADH activity; its low affinity for ethanol could explain the greater gender difference in first-pass metabolism with high rather than with low concentrations of imbibed alcohol. Alcohol gastric emptying was 42% slower and hepatic oxidation was 10% higher in women. A 7.3% smaller volume of alcohol distribution contributed to the higher ethanol levels in women, but it did not account for the route-dependent effects. CONCLUSIONS: The gender difference in alcohol levels is due mainly to a smaller gastric metabolism in females (because of a significantly lesser activity of chi-ADH), rather than to differences in gastric emptying or in hepatic oxidation of ethanol. The concentration-dependency of these effects may explain earlier discrepancies. The combined pharmacokinetic differences may increase the vulnerability of women to the effects of ethanol.     

The “First Hit” Toward Alcohol Reinforcement: Role of Ethanol Metabolites

This review analyzes literature that describes the behavioral effects of 2 metabolites of ethanol (EtOH): acetaldehyde and salsolinol (a condensation product of acetaldehyde and dopamine) generated in the brain. These metabolites are self‐administered into specific brain areas by animals, showing strong reinforcing effects. A wealth of evidence shows that EtOH, a drug consumed to attain millimolar concentrations, generates brain metabolites that are reinforcing at micromolar and nanomolar concentrations. Salsolinol administration leads to marked increases in voluntary EtOH intake, an effect inhibited by mu‐opioid receptor blockers. In animals that have ingested EtOH chronically, the maintenance of alcohol intake is no longer influenced by EtOH metabolites, as intake is taken over by other brain systems. However, after EtOH withdrawal brain acetaldehyde has a major role in promoting binge‐like drinking in the condition known as the “alcohol deprivation effect”; a condition seen in animals that have ingested alcohol chronically, are deprived of EtOH for extended periods, and are allowed EtOH re‐access. The review also analyzes the behavioral effects of acetate, a metabolite that enters the brain and is responsible for motor incoordination at low doses of EtOH. Also discussed are the paradoxical effects of systemic acetaldehyde. Overall, evidence strongly suggests that brain‐generated EtOH metabolites play a major role in the early (“first‐hit”) development of alcohol reinforcement and in the generation of relapse‐like drinking.     

Age-dependent changes in electroencephalographic responses to alcohol consumption in subjects with aldehyde dehydrogenase-2 genetic variations

BACKGROUND: We have recently reported that alcohol consumption resulted in a significant increase in alpha power of the EEGs in aldehyde dehydrogenase-2 (ALDH2)-normal (NN) subjects but not in ALDH2-deficient heterozygote (ND) subjects. The purpose of the present study was to investigate interactive effects of individual factors such as age and ALDH2 genotype on alcohol-induced EEG changes. METHODS: We examined EEG power spectral changes induced by 0.4 ml/kg of alcohol ingestion in 53 NN and 21 ND subjects of two different age groups: younger and older groups. Blood ethanol and acetaldehyde levels were also determined in 17 NN and 13 ND subjects in separate studies. RESULTS: Alcohol consumption markedly increased EEG power in the NN subjects of the older group, especially in theta and slow alpha power, whereas only slight increases were noted in fast alpha and beta power in the NN subjects of the younger group. However, no such differences between the two age groups were observed in the ND subjects. It should be noted that there were no differences in blood ethanol and acetaldehyde level at 30 min after alcohol ingestion between the different age groups in both genotypes. However, there was a significant increase in frequency of alcohol intake in the older group of both genotype groups. The multiple regression analysis indicated that both alcohol use habits and genotype, as well as aging, significantly modulated EEG changes after alcohol ingestion. CONCLUSIONS: The results suggest that both ALDH2 genotype and age as well as alcohol use habits modify alcohol sensitivity in the central nervous system, resulting in greater increases in EEG energy in response to alcohol intake in the older group of the NN subjects.     

Alcohol impairs protein synthesis and degradation in cultured skeletal muscle cells

BACKGROUND: Acute and chronic alcohol intoxication decreases skeletal muscle protein synthesis under in vivo conditions. We investigated whether ethanol (EtOH) and its major metabolites, acetaldehyde and acetate, can directly modulate protein balance under in vitro conditions. METHODS: Human myocytes were incubated with different doses of EtOH for varying periods of time (i.e., 4-72 hr). Alternatively, cells were incubated with acetaldehyde, acetate, insulin, insulin-like growth factor-I (IGF-I), or with a combination of EtOH plus insulin or IGF-I. Rates of protein synthesis or degradation were determined by 35S-methionine/cysteine incorporation into or release from cellular protein. RESULTS: A significant, 15% to 20%, decrease in basal protein synthesis was observed after 24 hr, but not at earlier time points, in response to 80 mM EtOH. Incubation of myocytes for 72 hr decreased synthesis in cells incubated with EtOH ranging between 60 and 120 mM. The ability of IGF-I or insulin to stimulate protein synthesis was impaired by 30% and 60%, respectively, in cells incubated with 80 mM EtOH for 72 hr. Exposure of cells to 200 microM acetaldehyde or 5 mM Na-acetate also decreased basal protein synthesis. In contrast, neither EtOH, acetaldehyde, nor acetate altered the basal rate of protein degradation. However, EtOH completely impaired the ability of insulin and IGF-I to inhibit proteolysis. Finally, EtOH did not impair IGF-I receptor autophosphorylation, but inhibited the ability of insulin to phosphorylate its own receptor. EtOH also did not alter the number of insulin or IGF-I receptors or the formation of insulin/IGF-I hybrid receptors. CONCLUSIONS: We have demonstrated that EtOH can directly inhibit muscle protein synthesis under in vitro conditions. Neither EtOH nor its metabolites altered basal protein degradation, although EtOH did compromise the ability of both insulin and IGF-I to slow proteolysis. This impairment seems to be mediated by different defects in signal transduction.     

High intracolonic acetaldehyde values produced by a bacteriocolonic pathway for ethanol oxidation in piglets.

BACKGROUND: Human colonic contents and many colonic microbes produce considerable amounts of acetaldehyde from ethanol in vitro. AIMS: To examine in piglets if acetaldehyde is produced in the colon also in vivo, and if so, what is the fate of intracolonically formed acetaldehyde. ANIMALS: Seventeen native, non-fasted female piglets (20-25 kg) were used. METHODS: Six piglets received either 1.5 g/kg bw or 2.5 g/kg bw of ethanol intravenously. In seven piglets, 0.7 g or 1.75 g of ethanol/kg bw was administered intravenously, followed by a subsequent intragastric ethanol infusion of 1.8 g/kg bw and 4.5 g/kg bw, respectively. The samples of colonic contents for the assessment of ethanol and acetaldehyde concentrations were obtained up to seven hours. In four additional piglets, the intracolonic values of ethanol, acetaldehyde, and acetate were observed for 60 minutes after an intracolonic infusion of acetaldehyde solution. RESULTS: A raised intracolonic, endogenous acetaldehyde concentration (mean (SEM); 36 (9) microM) was found in all piglets before ethanol infusion. After the infusion of ethanol, intracolonic ethanol and acetaldehyde values increased in parallel, reaching the peak values 57 (4) mM of ethanol and 271 (20) microM of acetaldehyde in the group that received the highest dose of ethanol. A positive correlation (r = 0.45; p < 0.001) was found between intracolonic ethanol and acetaldehyde values. Acetaldehyde administered intracolonically was mainly metabolised to acetate but also to ethanol in the colon. CONCLUSIONS: Significant endogenous intracolonic acetaldehyde values can be found in the normal porcine colon. Furthermore, our results suggest the existence of a bacteriocolonic pathway for ethanol oxidation. Increased amounts of acetaldehyde are formed intracolonically from ingested ethanol by this pathway.     

Determinants of Ethanol and Acetaldehyde Metabolism in Chronic Alcoholics

We have studied the factors determining the rate of ethanol and acetaldehyde metabolism in a group of 25 alcoholics with varying degrees of liver lesion (from normal liver to cirrhosis) and in six nonalcoholic cirrhotics. In alcoholics the ethanol metabolic rate was related to hepatic function, estimated either by the aminopyrine breath test ( r = 0.70, p < 0.001) or the indocyanine green clearance ( r = 0.76, p < 0.01), and was independent of the activity of hepatic alcohol dehydrogenase and hepatic blood flow. In nonalcoholic cirrhotics blood acetaldehyde was always below the detection limit (0.5 μM), but elevated levels were found in 14 out of the 25 alcoholics. Alcoholics with elevated blood acetaldehyde showed a significantly higher ethanol metabolic rate than alcoholics with undetectable acetaldehyde (120 ± 17 mg/kg/hr vs 104 ± 11 mg/kg/hr, p < 0.02), but no differences were observed in the activities of alcohol and aldehyde dehydrogenases. Peak blood acetaldehyde levels were directly related to the ethanol metabolic rate ( r = 0.48, p < 0.02), but not to activities of hepatic alcohol or aldehyde dehydrogenases. These results indicate that in chronic alcoholics the main determinant of the ethanol metabolic rate is hepatic function, while the rise of blood acetaldehyde is mainly dependent on the ethanol metabolic rate. Alcohol and aldehyde dehydrogenase activities do not seem to be rate-limiting factors in the oxidation of ethanol or acetaldehyde.     

Inhibition of alcohol dehydrogenase blocks enhanced Gi-protein expression following ethanol treatment in experimental hepatocellular carcinoma in vitro.

OBJECTIVE: Chronic alcohol abuse is one of the major contributors to the onset and progression of hepatocellular carcinoma (HCC). We have previously identified increased expression and function of inhibitory guanine nucleotide regulatory proteins (Gi-proteins) in primary human and animal models of HCC. Stimulation of Gi-proteins in HCC stimulates cell mitogenesis, an effect not observed in hepatocytes. The aim of this study was to determine the effect of ethanol and ethanol metabolism on Gi-protein expression in an experimental model of HCC. DESIGN: Pharmacological agents that inhibit alcohol metabolism were used in conjunction with ethanol or ethanol metabolites. We were also able to assess the relative contribution of alcohol and acetaldehyde, the major metabolite of alcohol, on Gi-protein expression in HCC and hepatocytes. METHODS: These studies used the rat hepatic tumorigenic H4IIE cell line in conjunction with isolated rat hepatocytes. Cells were cultured in vitro and exposed to ethanol, ethanol in the presence of an alcohol dehydrogenase (ADH) inhibitor, or acetaldehyde for varying lengths of time. Ethanol metabolism and changes in Gi-protein expression were subsequently determined by assay. RESULTS: Exposure to ethanol alone led to significant dose and time dependent increases in Gialpha1/2 and Gialpha3 protein and mRNA expression in HCC cells. In contrast, ethanol failed to alter Gialpha1/2, and only moderately affected Gialpha3 protein expression in isolated cultured hepatocytes. Pretreatment of HCC cells and hepatocytes with 4-methyl pyrazole (4-MP, 10 microm) significantly inhibited alcohol metabolism. Treatment of HCC cells with 4-MP inhibited changes in Gi-protein expression following exposure to ethanol (25 mm, 24 h). In addition, the increased expression of Gi-proteins observed after exposure to ethanol in HCC were mimicked by direct exposure of HCC cells to acetaldehyde in a dose and time dependent manner. CONCLUSIONS: These data suggest that alcohol metabolites, not alcohol, lead to increased Gi-protein expression in HCC in vitro. Ethanol and ethanol metabolites, in contrast, fail to significantly alter Gialpha1/2 protein expression in hepatocytes. These data may have significant implications in HCC progression in vivo.     

4-Methylpyrazole Decreases Salivary Acetaldehyde Levels in ALDH2-Deficient Subjects but Not in Subjects With Normal ALDH2

BACKGROUND: Carcinogenic acetaldehyde is produced from ethanol locally in the upper digestive tract via alcohol dehydrogenases (ADHs) of oral microbes, mucosal cells, and salivary glands. Acetaldehyde is further oxidized into less harmful acetate mainly by the aldehyde dehydrogenase-2 (ALDH2) enzyme. ALDH2-deficiency increases salivary acetaldehyde levels and the risk for upper digestive tract cancer in heavy alcohol drinkers. 4-methylpyrazole (4-MP) is an ADH-inhibitor which could reduce the local production of acetaldehyde from ethanol in the saliva. METHODS: Five ALDH2-deficient subjects and six subjects with normal ALDH2 ingested a moderate dose of alcohol (0.4 g/kg of body weight), whereafter their salivary acetaldehyde levels, heart rate, skin temperature, and blood pressure were followed for up to four hours. Blood acetaldehyde and ethanol levels were determined at 60 min. The experiment was repeated after a week. Two hours before the second study day, the volunteers received 4-MP, 10-15 mg/kg of body weight orally. RESULTS: Total ethanol elimination rate decreased with 4-MP by 38-46% in all subjects. 4-MP also reduced blood acetaldehyde levels and suppressed the cardiocirculatory responses of the ALDH2-deficient volunteers. In addition, salivary acetaldehyde production in ALDH2-deficient subjects was significantly reduced when correlated with salivary ethanol levels. On the contrary, 4-MP did not have any effect on salivary or blood acetaldehyde levels in subjects with normal ALDH2. CONCLUSIONS: A single dose of 4-MP before ethanol ingestion reduces ethanol elimination rate, the flushing reaction, and both blood and salivary acetaldehyde levels in ALDH2-deficient subjects but not in subjects with the normal ALDH2 genotype. These results suggest that the role of oral mucosal and glandular ADHs in salivary acetaldehyde production is minimal and support earlier findings indicating that salivary acetaldehyde production is mainly of microbial origin in subjects with normal ALDH2.     

Determinants of Blood Acetaldehyde Level during Ethanol Oxidation in Chronic Alcoholics

We analyzed the blood alcohol and acetaldehyde concentrations in nine alcoholics and four healthy nonalcoholic controls during and after an intravenous infusion of a high and a low dose of alcohol. In the alcoholics, the mean rates of plasma ethanol disappearance were significantly higher than in nonalcoholic controls. In the control subjects, the blood acetaldehyde levels were, in general, below the detection limit (less than 0.5 microM), but in sharp contrast to this, an elevated blood acetaldehyde during ethanol infusion was found in 6/9 alcoholics. Peak blood acetaldehyde values were higher after the high than the low dose of alcohol. Fructose infusion significantly enhanced the rate of plasma ethanol disappearance both in controls and in alcoholics, and this was usually associated with a significant elevation of blood acetaldehyde level. The maximal specific activities (expressed as milliunits/mg og protein) of alcohol, lactate, and aldehyde dehydrogenases in liver were significantly lower in alcoholics than in controls. Even more importantly, the peak blood acetaldehyde correlated negatively with the activity of hepatic "low-Km" aldehyde dehydrogenase. Our results suggest that the main reason for blood acetaldehyde elevation seen in these chronic alcoholics is their impaired capacity to metabolize acetaldehyde. This may be further accentuated by the increased rate of ethanol oxidation.     

Noncompetitive-Like Inhibition of Ethanol Elimination by Cyanamide Treatment: Pharmacokinetic Study

The effect of acetaldehyde accumulation of ethanol elimination is of interest in medico-legal practice in Japan. We examined the pharmacokinetic mechanism of the inhibition of ethanol metabolism by cyanamide, an inhibitor of mitochondrial aldehyde dehydrogenase. An ethanol solution (0.25-2.0 g/kg body weight) was injected intravenously into male rabbits with or without administration of cyanamide. Cyanamide was injected intraperitoneally (25 mg/kg body weight) to the cyanamide-treated group 2 hr before ethanol injection. Blood ethanol and acetaldehyde concentrations were measured periodically by head-space gas chromatography. The MULTI(RUNGE) computer program was applied for the pharmacokinetic analysis. One- or two-compartment open models with Michaelis-Menten elimination kinetics were used for simultaneous multi-line fitting. The ethanol elimination rate decreased by cyanamide treatment. The border-point concentration between pseudolinear and curvilinear phases was not affected by cyanamide treatment. The estimated V_max value decreased by cyanamide treatment, whereas the K_m value did not change. Our results correspond to a noncompetitive-like inhibition of ethanol metabolism. K_m is related to the border point between pseudolinear and curvilinear phases. Thus, our findings in the blood ethanol concentration-time curve suggest adequate curve-fitting. The product, or competitive, inhibition of alcohol dehydrogenase by acetaldehyde had been reported in enzymological study. The pharmacokinetic manner of inhibition in vivo was different from the enzymologic mechanism in vitro. Other metabolic factors related to ethanol metabolism are thought to be more important than acetaldehyde accumulation itself.     

Michaelis-Menten Elimination Kinetics of Acetate in Rabbit

BACKGROUND: Acetate redistribution from hepatic to peripheral tissues was reported during ethanol metabolism when saturating conditions were reached for acetate metabolism. Because this redistribution cannot be clarified by linear kinetics, elimination kinetics of acetate was studied in the rabbit. METHODS: A sodium-acetate solution in physiological saline (0.5 and 1.0 g/kg of body weight) was injected as an intravenous bolus. The blood acetate profile was measured by headspace gas chromatography. RESULTS: Blood acetate disappeared rapidly. Statistical moment analysis of the blood acetate profiles showed that the normalized area under the curve and the mean residence time increased with an increasing dose amount. These increases suggested a capacity-limited elimination of acetate. Simultaneous multilines fitting after two acetate doses was used to estimate the pharmacokinetic model by the application of minimum Akaike's information criterion estimation. As a result, the blood acetate concentration-time curve was best described by a two-compartment open model with Michaelis-Menten elimination kinetics. The Vmax value was approximately two times larger than that of ethanol obtained by using the same compartment model. The Km value (1.5 mM) was almost the same as that of ethanol and corresponded to blood acetate levels during ethanol oxidation that had been reported to be approximately 2 mM. CONCLUSION: The elimination of acetate obeys nonlinear kinetics, which can clarify the saturation of acetate metabolism.     

Assays for Acetaldehyde-Derived Adducts in Blood Proteins Based on Antibodies Against Acetaldehyde/Lipoprotein Condensates

BACKGROUND: Acetaldehyde-derived protein condensates (adducts) have been suggested as promising biological markers of alcohol abuse because they represent actual metabolites of ethanol. However, the detection of such condensates in vivo has been hampered by a lack of sensitive and specific methods. METHODS: To develop new approaches for the detection of acetaldehyde adducts, we have raised antibodies against condensates with acetaldehyde and lipoproteins, which have previously been shown to be readily modified by acetaldehyde in vitro. The characteristics of these antibodies were compared with those raised against bovine serum albumin/acetaldehyde adduct and against other types of lipoprotein modifications, as induced by malondialdehyde, oxidation, and acetylation. RESULTS: The antibodies raised against low-density lipoprotein (LDL)/acetaldehyde, very low density lipoprotein (VLDL)/acetaldehyde, and bovine serum albumin/acetaldehyde all reacted with protein adducts generated at physiologically relevant concentrations of acetaldehyde in vitro, whereas the antibodies raised against malondialdehyde/LDL, oxidized LDL, or acetylated LDL were not found to cross-react with the acetaldehyde-derived adducts. In assays for acetaldehyde adducts from erythrocyte and serum proteins of patients with excessive ethanol consumption (n = 32) and healthy control individuals (n = 22), the antibody prepared against the acetaldehyde/VLDL condensate was found to provide the most effective detection of acetaldehyde adducts in vivo. CONCLUSIONS: Current data indicate that acetaldehyde generates immunogenic adducts with lipoproteins in vivo. Antibodies raised against the VLDL/acetaldehyde may provide a basis for new diagnostic assays to examine excessive alcohol consumption.     

Acetaldehyde Exposure Causes Growth Inhibition in a Chinese Hamster Ovary Cell Line That Expresses Alcohol Dehydrogenase

Chronic ethanol exposure causes many pathophysiological changes in cellular function due to ethanol itself and/or the effects of its metabolism (i.e., generation of acetaldehyde and redox equivalents). However, the role of each of these effects remains controversial. To address these questions, we have developed a cell line that expresses alcohol dehydrogenase. This cell line permits separate examination of the effects of ethanol and its metabolite acetaldehyde on cell function. An expression vector for the mouse liver alcohol dehydrogenase was constructed and transfected into Chinese hamster ovary cells. Cells expressing alcohol dehydrogenase were identified by screening with allyl alcohol, which is metabolized by alcohol dehydrogenase to the toxic aldehyde acrolein. A number of cell lines were identified that expressed alcohol dehydrogenase. A-10 cells were selected for further study because of their high sensitivity to allyl alcohol, suggesting a high level of alcohol dehydrogenase expression. These cells expressed a mRNA that hybridizes with the alcohol dehydrogenase cDNA and had an alcohol dehydrogenase activity comparable to murine liver. When cultures of these cells were exposed to ethanol, acetaldehyde was detected in both the medium and cells. The acetaldehyde concentration in the medium remained constant for at least 1 week in culture and was a function of the added ethanol concentration. Chronic exposure of A-10 cells to ethanol resulted in a dose-dependent reduction in the number of cells that accumulated over 7 days. Ethanol-treated cells remained viable, and growth inhibition was reversible. Growth inhibition was blocked by the alcohol dehydrogenase inhibitor 4-methylpyrazole, suggesting that acetaldehyde and not ethanol was responsible for growth inhibition in these cells.     

Polymorphisms of Alcohol Dehydrogenase‐1B and Aldehyde Dehydrogenase‐2 and the Blood and Salivary Ethanol and Acetaldehyde Concentrations of Japanese Alcoholic Men

Background: The effects of genetic polymorphism of aldehyde dehydrogenase‐2 (ALDH2) on alcohol metabolism are striking in nonalcoholics, and the effects of genetic polymorphism of alcohol dehydrogenase‐1B (ADH1B) are modest at most, whereas genetic polymorphisms of both strongly affect the susceptibility to alcoholism and upper aerodigestive tract (UADT) cancer of drinkers. Methods: We evaluated associations between ADH1B/ADH1C/ALDH2 genotypes and the blood and salivary ethanol and acetaldehyde levels of 168 Japanese alcoholic men who came to our hospital for the first time in the morning and had been drinking until the day before. Results: The ethanol levels in their blood and saliva were similar, but the acetaldehyde levels in their saliva were much higher than in their blood, probably because of acetaldehyde production by oral bacteria. Blood and salivary ethanol and acetaldehyde levels were both significantly higher in the subjects with the less active ADH1B*1/*1 genotype than in the ADH1B*2 carriers, but none of the levels differed according to ALDH2 genotype. Significant linkage disequilibrium was detected between the ADH1B and ADH1C genotypes, but ADH1C genotype did not affect the blood or salivary ethanol or acetaldehyde levels. High blood acetaldehyde levels were found even in the active ALDH2*1/*1 alcoholics, which were comparable with the levels of the inactive heterozygous ALDH2*1/*2 alcoholics with less active ADH1B*1/*1. The slope of the increase in blood acetaldehyde level as the blood ethanol level increased was significantly steeper in alcoholics with inactive heterozygous ALDH2*1/*2 plus ADH1B*2 allele than with any other genotype combinations, but the slopes of the increase in salivary acetaldehyde level as the salivary ethanol level increased did not differ between the groups of subjects with any combinations of ALDH2 and ADH1B genotypes. Conclusions: The ADH1B/ALDH2 genotype affected the blood and salivary ethanol and acetaldehyde levels of nonabstinent alcoholics in a different manner from nonalcoholics, and clear effects of ADH1B genotype and less clear effects of ALDH2 were observed in the alcoholics. Alterations in alcohol metabolism as a result of alcoholism may modify the gene effects, and these findings provide some clues in regard to associations between the genotypes and the risks of alcoholism and UADT cancer.     

Ethanol, acetaldehyde, and estradiol affect growth and differentiation of rhesus monkey embryonic stem cells

Background: The timing of the origins of fetal alcohol syndrome has been difficult to determine, in part because of the challenge associated with in vivo studies of the peri‐implantation stage of embryonic development. Because embryonic stem cells (ESCs) are derived from blastocyst stage embryos, they are used as a model for early embryo development. Methods: Rhesus monkey ESC lines (ORMES‐6 and ORMES‐7) were treated with 0, 0.01, 0.1, or 1.0% ethanol, 1.0% ethanol with estradiol, or 0.00025% acetaldehyde with or without estradiol for 4 weeks. Results: Although control ESCs remained unchanged, abnormal morphology of ESCs in the ethanol and acetaldehyde treatment groups was observed before 2 weeks of treatment. Immunofluorescence staining of key pluripotency markers (TRA‐1‐81 and alkaline phosphatase) indicated a loss of ESC pluripotency in the 1.0% ethanol group. ORMES‐7 was more sensitive to effects of ethanol than ORMES‐6. Conclusions: Estradiol appeared to increase sensitivity to ethanol in the ORMES‐6 and ORMES‐7 cell line. The morphological changes and labeling for pluripotency, proliferation, and apoptosis demonstrated that how ethanol affects these early cells that develop in culture, their differentiation state in particular. The effects of ethanol may be mediated in part through metabolic pathways regulating acetaldehyde formation, and while potentially accentuated by estradiol in some individuals, how remains to be determined.     

A Critical Evaluation of Influence of Ethanol and Diet on Salsolinol Enantiomers in Humans and Rats

Background: (R/S)‐Salsolinol (SAL), a condensation product of dopamine (DA) with acetaldehyde, has been speculated to have a role in the etiology of alcoholism. Earlier studies have shown the presence of SAL in biological fluids and postmortem brains from both alcoholics and nonalcoholics. However, the involvement of SAL in alcoholism has been controversial over several decades, since the reported SAL levels and their changes after ethanol exposure were not consistent, possibly due to inadequate analytical procedures and confounding factors such as diet and genetic predisposition. Using a newly developed mass spectrometric method to analyze SAL stereoisomers, we evaluated the contribution of ethanol, diet, and genetic background to SAL levels as well as its enantiomeric distribution. Methods: Simultaneous measurement of SAL enantiomers and DA were achieved by high performance liquid chromatography‐tandem mass spectrometry (HPLC/MS/MS). Plasma samples were collected from human subjects before and after banana (a food rich in SAL) intake, and during ethanol infusion. Rat plasma and brain samples were collected at various time points after the administration of SAL or banana by gavage. The brain parts including nucleus accumbens (NAC) and striatum (STR) were obtained from alcohol‐non‐preferring (NP) or alcohol‐preferring (P) rats as well as P‐rats which had a free access to ethanol (P‐EtOH). Results: Plasma SAL levels were increased significantly after banana intake in humans. Consistently, administration of banana to rats also resulted in a drastic increase of plasma SAL levels, whereas brain SAL levels remained unaltered. Acute ethanol infusion did not change SAL levels or R/S ratio in plasma from healthy humans. The levels of both SAL isomers and DA were significantly lower in the NAC of P rats in comparison to NP rats. The SAL levels in NAC of P rats remained unchanged after chronic free‐choice ethanol drinking. There were decreasing trends of SAL in STR and DA in both brain regions. No changes in enantiomeric ratio were observed after acute or chronic ethanol exposure. Conclusions: SAL from dietary sources is the major contributor to plasma SAL levels. No significant changes of SAL plasma levels or enantiomeric distribution after acute or chronic ethanol exposure suggest that SAL may not be a biomarker for ethanol drinking. Significantly lower SAL and DA levels observed in NAC of P rats may be associated with innate alcohol preference.     

Ethanol, acetaldehyde, acetate, and lactate levels after alcohol intake in white men and women: effect of 4-methylpyrazole

BACKGROUND: 4-Methylpyrazole (4-MP), a selective inhibitor of alcohol dehydrogenase (ADH), recently has been approved for clinical use in humans. The objective was to evaluate the use of 4-MP in human alcohol research and to study the effect of 4-MP on various parameters of alcohol metabolism during alcohol intoxication. METHODS: 4-MP (10-15 mg/kg orally) or placebo was given in double-blind fashion to 22 premenopausal women, 12 of whom were using oral contraceptives, and 13 men followed by intake of alcohol (0.5 g/kg orally) or placebo. RESULTS: A 30% to 40% decrease in the ethanol elimination rate was observed in the different groups during pretreatment with 4-MP. The alcohol-induced increase in plasma acetate was partially inhibited by 4-MP. A significant positive correlation was observed between the effect of 4-MP on the alcohol-induced lactate and acetate elevations. The acetaldehyde was nondetectable (<1 micromol/liter) in the peripheral venous blood during alcohol intoxication in both women and men. During alcohol intoxication, a decrease in breath acetaldehyde was found with 4-MP pretreatment in women but not in men. CONCLUSION: The alcohol-induced elevation in blood acetate level is caused, in part, by ADH-mediated ethanol oxidation. Although no evidence was found for measurable acetaldehyde levels in the peripheral venous blood during alcohol intoxication, the effect of 4-MP on breath acetaldehyde in women supports the view that ADH-mediated acetaldehyde elevations reflected in the airways, but too low to be detected in the peripheral venous blood, may occur in women during alcohol intoxication in the present experimental conditions.     

Multi-Enzyme Catalyzed Rapid Ethanol Lowering in Vitro

Ethanol was oxidized to acetate by an enzyme system using yeast alcohol dehydrogenase (YADH), yeast aldehyde dehydrogenase (YALDH), and lactic dehydrogenase (LDH) recycling NAD in two model duodenal fluids and in canine duodenal aspirate in vitro. Sufficient enzyme activities were maintained to convert as much as 34% of the original ethanol to acetate with negligible acetaldehyde accumulation.