DEUTERIUM AND TRITIUM ISOTOPE EFFECTS ON THE MICROSOMAL N-DEMETHYLATION OF ETHYLMORPHINE

Ethylmorphine (EM) and trideuteriomethylnorethylmorphine (EM-CD3) were incubated with rat liver microsomes and an NADPH-generating system in parallel experiments. N-Demethyla-tion was determined by measuring the formaldehyde formed. After 10 mm at 37#{176}C.,the average deuterium isotope effect (k’Vk#{176})was 1.25. Similar small effects were found when incubations were terminated after 1. 2. 4. 8. and 16 mm. The kH/k) 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 VX/V.X 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 be-tween 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 tne overall rate of N-demethylation