The long-term objective of this research project is to elucidate the molecular basis of individual differences in alcohol elimination. The hypothesis is that genetic differences in the enzymes of alcohol metabolism account for a substantial part of this variance and may, in part underlie the observed individual differences in the physiological, psychological and pathological consequences of ethanol consumption. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are the principal enzymes responsible for ethanol metabolism in the liver. There are 5 structural genes for ADH in humans, and variant alleles for one of them, ADH2 , yield isozymes with strikingly different catalytic properties: the beta 2 (subunit)- containing isozymes seen in 85% of Asians and the beta 3 -containing isozymes seen in 27% of African-Americans have higher Km's and Vmax's for ethanol than do the beta 1-containing isozymes found in all three population groups. Since it is now possible to genotype the ADH loci by use of leukocyte DNA samples, it is proposed to compare the pharmacokinetics of ethanol elimination in African-American subjects with the following genotypes: ADH2(1)/ADH2(1) (beta 1 subunits only), ADH2(1)/ADH2(3) (beta 1 and beta 3) and ADH2(3)/ADH2(3) (beta 3 only.) Similarly, 3 groups of Asian subjects with genotypes ADH2(1)/ADH2(1), ADH2(1)/ADH2(2) (beta 1 and beta 2), and ADH2(2)/ADH2(2) (beta 2 only) will be compared. Alcohol elimination rates after oral ethanol administration (0.8 - 0.6 g/kg as moderate dose and 1.0 - 0.9 g/kg as high dose) will be measured. In other studies, kinetic characterization of the different human liver ADH isozymes will be further pursued to discern the effects of different amino acid substitutions. Stopped-flow kinetics and chemical modification of active site His, Arg and Cys residues will be performed. The high-Km-for-ethanol ADH form in human stomach (sigma-ADH), which appears to be similar to the pi-ADH form in liver, will be isolated and characterized. cDNA libraries will be prepared from human stomachs and the sigma-ADH cDNA will be cloned and sequenced. The sigma-ADH gene will be cloned and compared with other ADH genes. Finally, the formation and catalytic properties of the tetrameric human mitochondrial ALDH (ALDH2) formed from the mixing of active subunits (product of the ALDH2(1) allele) and the inactive subunits (product of the ALDH2(2) allele) will be studied. The amount of the different ALDH2 heterotetramers formed in livers with the heterozygous ALDH2(1)/ALDH2(2) genotype will be measured and their activity and stability will be determined.
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