Modeling of Human Hepatic and Gastrointestinal Ethanol Metabolism with Kinetic-Mechanism-Based Full-Rate Equations of the Component Alcohol Dehydrogenase Isozymes and Allozymes.

Alcohol dehydrogenase (ADH) is the principal enzyme responsible for the metabolism of ethanol. Human ADH constitutes a complex family of isozymes and allozymes with striking variation in kinetic properties and tissue distribution. The liver and the gastrointestinal tract are the major sites for first-pass metabolism (FPM). The quantitative contributions of ADH isozymes and ethnically distinct allozymes to cellular ethanol metabolism remain poorly understood. To address this issue, kinetic mechanism and the steady-state full-rate equations for recombinant human class I ADH1A, ADH1B (including allozymes ADH1B1, ADH1B2, and ADH1B3), ADH1C (including allozymes ADH1C1 and ADH1C2), class II ADH2, and class IV ADH4 were determined by initial velocity, product inhibition, and dead-end inhibition experiments in 0.1 M sodium phosphate at pH 7.5 and 25 °C. Models of the hepatic and gastrointestinal metabolisms of ethanol were constructed by linear combination of the numerical full-rate equations of the component isozymes and allozymes in target organs. The organ simulations indicate that in homozygous ADH1B*1/*1 livers, a representative genotype among ethnically distinct populations due to high prevalence of the allele, major contributors at 1 to 10 mM ethanol are ADH1B1 (45% to 24%) and the ADH1C allozymes (54% to 40%). The simulated activities at 1 to 50 mM ethanol for the gastrointestinal tract (total mucosae of ADH1C*1/*1-ADH4 stomach and the ADH1C*1/*1-ADH2 duodenum and jejunum) account for 0.68%-0.76% of that for the ADH1B*1/*1-ADH1C*1/*1 liver, suggesting gastrointestinal tract plays a relatively minor role in the human FPM of ethanol. Based on the flow-limited sinusoidal perfusion model, the simulated hepatic Kmapp, Vmaxapp, and Ci at a 95% clearance of ethanol for ADH1B*1/*1-ADH1C*1/*1 livers are compatible to that documented in hepatic vein catheterization and pharmacokinetic studies with humans that controlled for the genotypes. The model simulations suggest that slightly higher or similar ethanol elimination rates for ADH1B*2/*2 and ADH1B*3/*3 individuals compared with those for ADH1B*1/*1 individuals may result from higher hepatocellular acetaldehyde.

Ethanol oxidation and the inhibition by drugs in human liver, stomach and small intestine: Quantitative assessment with numerical organ modeling of alcohol dehydrogenase isozymes.

Alcohol dehydrogenase (ADH) is the principal enzyme responsible for metabolism of ethanol. Human ADH constitutes a complex isozyme family with striking variations in kinetic function and tissue distribution. Liver and gastrointestinal tract are the major sites for first-pass metabolism (FPM). Their relative contributions to alcohol FPM and degrees of the inhibitions by aspirin and its metabolite salicylate, acetaminophen and cimetidine remain controversial. To address this issue, mathematical organ modeling of ethanol-oxidizing activities in target tissues and that of the ethanol-drug interactions were constructed by linear combination of the corresponding numerical rate equations of tissue constituent ADH isozymes with the documented isozyme protein contents, kinetic parameters for ethanol oxidation and the drug inhibitions of ADH isozymes/allozymes that were determined in 0.1 M sodium phosphate at pH 7.5 and 25 °C containing 0.5 mM NAD(+). The organ simulations reveal that the ADH activities in mucosae of the stomach, duodenum and jejunum with ADH1C*1/*1 genotype are less than 1%, respectively, that of the ADH1B*1/*1-ADH1C*1/*1 liver at 1-200 mM ethanol, indicating that liver is major site of the FPM. The apparent hepatic KM and Vmax for ethanol oxidation are simulated to be 0.093 ± 0.019 mM and 4.0 ± 0.1 mmol/min, respectively. At 95% clearance in liver, the logarithmic average sinusoidal ethanol concentration is determined to be 0.80 mM in accordance with the flow-limited gradient perfusion model. The organ simulations indicate that higher therapeutic acetaminophen (0.5 mM) inhibits 16% of ADH1B*1/*1 hepatic ADH activity at 2-20 mM ethanol and that therapeutic salicylate (1.5 mM) inhibits 30-31% of the ADH1B*2/*2 activity, suggesting potential significant inhibitions of ethanol FPM in these allelotypes. The result provides systematic evaluations and predictions by computer simulation on potential ethanol FPM in target tissues and hepatic ethanol-drug interactions in the context of tissue ADH isozymes.

Ethanol-metabolizing activities and isozyme protein contents of alcohol and aldehyde dehydrogenases in human liver: phenotypic traits of the ADH1B*2 and ALDH2*2 variant gene alleles.

OBJECTIVE: Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are principal enzymes responsible for the metabolism of ethanol. East Asian populations are unique in that they carry both a prevalent ADH1B*2 and a dominant-negative ALDH2*2 allele. A systematic investigation of ethanol-metabolizing activities in normal livers correlated with the corresponding functional allelic variations and protein contents of the relevant isozymes in respective enzyme families has been lacking. MATERIALS AND METHODS: To obtain a reasonable sample size encompassing all possible genetic allelotypes of the ADH1B and ALDH2, 141 surgical liver specimens from adult Han Chinese were studied. Expression patterns and activities of ADH and ALDH were determined with stratification of the genetic phenotypes. Absolute protein contents as well as cellular localization of the activity and protein of ADH/ALDH isozymes were also investigated. RESULTS: The activities of ADH1B*1/*2 and ADH1B*2/*2 allelic phenotypes were 5-6-fold those of the ADH1B*1/*1, suggesting that ADH1B*2 allele-encoded subunits are dominant over expression of hepatic ADH activity. The activities of the ALDH2-active phenotype were 90% higher than those of the ALDH2-inactive phenotype. Sex and age did not significantly influence the hepatic ADH and ALDH activities with specified genetic phenotypes. The isozyme protein contents were as follows in decreasing order: ADH1, ADH2, ALDH1A1, ALDH2, and ADH3. Both ADH1, but not ADH2/3, and ALDH1A1/2 showed a preferential expression in perivenular hepatocytes. CONCLUSION: Functional correlations of ADH1B*2 and ALDH2*2 variant alleles in the liver provide a biochemical genetic basis suggesting their contribution toward variability in ethanol metabolism as well as susceptibility to alcoholism and alcohol-related diseases in East Asians.

Inhibition of human alcohol and aldehyde dehydrogenases by aspirin and salicylate: assessment of the effects on first-pass metabolism of ethanol.

Previous studies have reported that aspirin significantly reduced the first-pass metabolism (FPM) of ethanol in humans thereby increasing adverse effects of alcohol. The underlying causes, however, remain poorly understood. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), principal enzymes responsible for metabolism of ethanol, are complex enzyme families that exhibit functional polymorphisms among ethnic groups and distinct tissue distributions. We investigated the inhibition profiles by aspirin and its major metabolite salicylate of ethanol oxidation by recombinant human ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2, ADH2, and ADH4, and acetaldehyde oxidation by ALDH1A1 and ALDH2, at pH 7.5 and 0.5 mM NAD(+). Competitive inhibition pattern was found to be a predominant type among the ADHs and ALDHs studied, although noncompetitive and uncompetitive inhibitions were also detected in a few cases. The inhibition constants of salicylate for the ADHs and ALDHs were considerably lower than that of aspirin with the exception of ADH1A that can be ascribed to a substitution of Ala-93 at the bottom of substrate pocket as revealed by molecular docking experiments. Kinetic inhibition equation-based simulations show at higher therapeutic levels of blood plasma salicylate (1.5 mM) that the decrease of activities at 2-10 mM ethanol for ADH1A/ADH2 and ADH1B2/ADH1B3 are predicted to be 75-86% and 31-52%, respectively, and that the activity decline for ALDH1A1 and ALDH2 at 10-50 µM acetaldehyde to be 62-73%. Our findings suggest that salicylate may substantially inhibit hepatic FPM of alcohol at both the ADH and ALDH steps when concurrent intaking aspirin.

Oxidation of methanol, ethylene glycol, and isopropanol with human alcohol dehydrogenases and the inhibition by ethanol and 4-methylpyrazole.

Human alcohol dehydrogenases (ADHs) include multiple isozymes with broad substrate specificity and ethnic distinct allozymes. ADH catalyzes the rate-limiting step in metabolism of various primary and secondary aliphatic alcohols. The oxidation of common toxic alcohols, that is, methanol, ethylene glycol, and isopropanol by the human ADHs remains poorly understood. Kinetic studies were performed in 0.1M sodium phosphate buffer, at pH 7.5 and 25°C, containing 0.5 mM NAD(+) and varied concentrations of substrate. K(M) values for ethanol with recombinant human class I ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, and ADH1C2, and class II ADH2 and class IV ADH4 were determined to be in the range of 0.12-57 mM, for methanol to be 2.0-3500 mM, for ethylene glycol to be 4.3-2600mM, and for isopropanol to be 0.73-3400 mM. ADH1B3 appeared to be inactive toward ethylene glycol, and ADH2 and ADH4, inactive with methanol. The variations for V(max) for the toxic alcohols were much less than that of the K(M) across the ADH family. 4-Methylpyrazole (4MP) was a competitive inhibitor with respect to ethanol for ADH1A, ADH1B1, ADH1B2, ADH1C1 and ADH1C2, and a noncompetitive inhibitor for ADH1B3, ADH2 and ADH4, with the slope inhibition constants (K(is)) for the whole family being 0.062-960 µM and the intercept inhibition constants (K(ii)), 33-3000 µM. Computer simulation studies using inhibition equations in the presence of alternate substrate ethanol and of dead-end inhibitor 4MP with the determined corresponding kinetic parameters for ADH family, indicate that the oxidation of the toxic alcohols up to 50mM are largely inhibited by 20 mM ethanol or by 50 µM 4MP with some exceptions. The above findings provide an enzymological basis for clinical treatment of methanol and ethylene glycol poisoning by 4MP or ethanol with pharmacogenetic perspectives.