| Summary: | 博士 === 國防醫學院 === 生命科學研究所 === 89 === Alcohol dehydrogenase (ADH) is the major enzyme responsible for ethanol metabolism in mammals. The purpose of this dissertation is (a) to investigate human gastric ADH activity in relation to the expression pattern, gender and age, and to estimate the extent of first-pass metabolism of ethanol in the stomach and liver in humans and rats by simulation studies using substrate inhibition equations of ADHs which are expressed with a tissues-specific fashion, and (b) to investigate steady-state metabolism of ethanol in the rat liver using nonrecirculated perfusion technique and to evaluate factors which affect alcohol metabolic rate by simulation using ADH steady-state kinetic mechanism-based rate equation.
A total of 115 biopsy specimens of stomach mucosae were collected by gastroendoscopy from 70 men and 45 women aged between 20—79 which were approximately in even distribution among 10-year intervals. Expression patterns of ADH and aldehyde dehydrogenase (ALDH) were identified by agarose isoelectric focusing. Distribution of the phenotypes of ADH or ALDH was found no difference with respect to gender and age subgroups. ADH activities at 33 mM or 500 mM ethanol (pH 7.5) were found no difference between men and women or among age subgroups. ALDH activities at 200 μM or 20 mM acetaldehyde were found no difference with respect to gender and age subgroups. Linear regression analyses indicated that there was no significant correlation between ADH or ALDH activity and age in men and women. These results suggest that gastric ADH and ALDH activity do not significantly contribute to the difference of first-pass metabolism of alcohol related to gender or age. Simulation studies using kinetic mechanism-based rate equations of ADHs which are expressed in the liver or stomach indicate that liver is the major organ responsible for first-pass metabolism of alcohol in humans, and in rats the major metabolism organ is stomach instead of liver.
Class I ADH was isolated from rat liver via DE-52 ion-exchange and CapGapp affinity chromatographic procedures to apparent homogeneity as judged by SDS-PAGE. The subunit molecular weight was determined to be 40,000 Da. Three activity bands of the rat class I ADH, revealed on polyacrylamide iselectric focusing gel, were resolved and isolated by Mono S FPLC. Close similarity of the kinetic properties of the 3 class I ADH forms suggests that they belong to the same enzyme, and two of them may be generated from post-translational modifications or artifact of the purification procedures. Results of initial velocity, production inhibition, and dead-end inhibition studies indicate that class I rat ADH is consistent with Theorell-Chance mechanism with substrate inhibition at high ethanol concentrations (> 5 mM).
Non-recirculated perfusion studies of rat liver demonstrate that class I ADH is the major enzyme responsible for steady-state metabolism of alcohol by following findings: (i) substrate inhibition of ethanol, (ii) competitive inhibition by 4-methylpyrazole, and (iii) uncompetitive inhibition by isobutyramide, which all were in well agreement with the results predicted from simulation studies using kinetic-mechanism based equations of rat liver ADH. Non-ADH pathways involved in the metabolism of ethanol in the rat liver were estimated to be less than 5% by 4-methylpyrazole inhibition studies. Simulation results indicate that the major factors that affect alcohol metabolic rate in the rat liver may vary with ethanol concentrations, i.e. (a) subsaturation of substrates (ethanol and NAD) and product (NADH) inhibition are the major factors as ethanol concentrations are below 2 mM, (b) substrate (ethanol and NAD) subsaturation, product (NADH) inhibition, and substrate (ethanol) inhibition are the major factors as ethanol concentrations fall between 2—10 mM, and (c) substrate (ethanol) inhibition becomes a main factor as ethanol concentrations approach 100 mM.
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