Abstract

A radioassay for in vitro ethylmorphine N-demethylase activity has been developed which combines high sensitivity and convenience. Aliquots from incubation mixtures containing rat liver microsomes, a reduced nicotinamide adenine dinucleotide phosphate-genenrating system, and N-methyl-³H-ethylmorphine are passed through columns of XAD-2 resin. This treatment efficiently removes the labeled substrate permitting enzymatically released tritium to be measured in the column effluent as ³HCHO and ³H₂O. That tritium release is a valid measure of demethylase activity was established by comparing levels of metabolism obtained by both XAD-2 and colorimetric assays. A straight line and the expected K_m of 0.24 mM were obtained with radioassay data on a standard double-reciprocal plot. Radioactivity release was linear with microsomal protein concentratioim. The XAD-2 assay was found to be about 30-fold more sensitive than colorimetric methods, since a level of metabolism corresponding to the formation of 0.41 nmol of HCHO per ml could be measured. Lowering time background radioactivity by derivatizing ³HCHO with dimedon and extracting the derivative with benzene-hexane further increased the sensitivity to a factor of 75 over colonimetric methods. In this way, the metabolism of 10 µg of microsomal protein per ml of incubate could be measured, corresponding to time formation of only 0.16 nmol of HCHO per ml. The use of very low ³H-ethylmorphine concentrations reduced background levels and allowed us to assay 0.040 nmoi of HCHO per ml.