TY - JOUR T1 - DEUTERIUM AND TRITIUM ISOTOPE EFFECTS ON THE MICROSOMAL N-DEMETHYLATION OF ETHYLMORPHINE JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 577 LP - 582 VL - 2 IS - 6 AU - JOHN A. THOMPSON AU - JORDAN L. HOLTZMAN Y1 - 1974/11/01 UR - http://dmd.aspetjournals.org/content/2/6/577.abstract N2 - Ethylmorphine (EM) and trideuteriomethylnorethylmorphine (EM-CD3) were incubated with rat liver microsomes and an NADPH-generating system in parallel experiments. N-Demethylation was determined by measuring the formaldehyde formed. After 10 min at 37°C, the average deuterium isotope effect (kH/kD) was 1.25. Similar small effects were found when incubations were terminated after 1, 2, 4, 8, and 16 min. The kH/kD did not vary when the incubations were buffered over the pH range from 5.6 to 8.4. Standard double-reciprocal plots of EM and EM-CD3 concentration against the rate of formaldehyde formation yielded a VmaxH/VmaxD of 1.26 and identical KM values of 0.21 mM. Oxygen uptake experiments in the presence of EM and EM-CD3 demonstrated that deuterium substitution had no effect on this rate. A tritium isotope effect of 2.49 was calculated by incubating N-methyl-tritiated EM with microsomes, removing unreacted substrate from solution, and determining the distribution of tritium between water and formaldehyde. These data, together with that from previous experiments, demonstrate that the small isotope effects found with deuterated and tritiated EM are genuine rate effects and that two steps contribute significantly to the overall rate of N-demethylation. These are the cleavage of the C—H bond and the donation of a second electron to the proposed cytochrome P-450-substrate-superoxide complex. Copyright © 1974 by The American Society for Pharmacology and Experimental Therapeutics ER -