Abstract

Erythromycin N-demethylation is catalyzed by cytochrome P4503A isozymes. By using [14C]methyl-labeled erythromycin, we were able to develop a N-demethylation assay that is more sensitive and specific than the colorimetric detection of formaldehyde formation. The increased sensitivity allows the use of very low substrate concentration with good sensitivity, 1 microM compared with 400 microM for the colorimetric assay. This 1 microM concentration is within pharmacological blood levels of erythromycin. Using this assay, we detected a high-affinity erythromycin N-demethylase in liver microsomes from untreated adult female rats that was previously unknown. This low KM activity could be inhibited by polyclonal anti-P4503A1 or P4503A2 antibodies to 95%, and these antibodies also detected a band in these microsomes on Western blots that had the same molecular weight (51 kDa) as cytochromes P4503A1/3A2. Monoclonal antibodies specific for P4503A1 or P4503A2, however, did not react with this band. No inhibitory effect was observed with monoclonal antibody P124, which inhibited the erythromycin N-demethylation both in liver microsomes from untreated adult males (P4503A2) and dexamethasone-pretreated adult females (P4503A1). Alternative P4503A substrates (testosterone, troleandomycin, cortisol, corticosterone, cyclosporin A, and 17 alpha-ethinylestradiol) inhibited erythromycin N-demethylation catalyzed by liver microsomes from untreated male, untreated female, and dexamethasone-pretreated female rats, whereas digitoxin and theophylline had no inhibitory effects. Put together, these data suggest that this demethylase in liver microsomes of untreated female rats is not P4503A1 or P4503A2, but P4503A related.