ERYTHROMYCIN AS A SPECIFIC SUBSTRATE FOR CYTOCHROME P4503A ISOZYMES AND IDENTIFICATION OF A HIGH-AFFINITY ERYTHROMYCIN N-DEMETHYLASE IN ADULT FEMALE RATS

Erythromycin N-demethylation is catalyzed by cytochrome P4503A isozyme s, By using [C-14]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 sensit ivity allows the use of very low substrate concentration with good sen sitivity, 1 mu M compared with 400 mu M for the colorimetric assay, Th is 1 mu M concentration is within pharmacological blood levels of eryt hromycin. 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 K-M activity could be inhibited by p olyclonal anti-P4503A1 or P4503A2 antibodies to 95%, and these antibod ies also detected a band in these microsomes on Western blots that had the same molecular weight (51 kDa) as cytochromes P4503A1/3A2. Monocl onal antibodies specific for P4503A1 or P4503A2, however, did not reac t with this band. No inhibitory effect was observed with monoclonal an tibody P124, which inhibited the erythromycin N-demethylation both in liver microsomes from untreated adult males (P4503A2) and dexamethason e-pretreated adult females (P4503A1), Alternative P4503A substrates (t estosterone, troleandomycin, cortisol, corticosterone, cyclosporin A, and 17 alpha-ethinylestradiol) inhibited erythromycin N-demethylation catalyzed by liver microsomes from untreated male, untreated female, a nd dexamethasone-pretreated female rats, whereas digitoxin and theophy lline 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.