HPLC analysis of 4-hydroxytamoxifen glucuronides formed by wild-type UGT1A4-overexpressing cell microsomes. UGT1A424Pro/48Leu-overexpressing cell microsomes (10 μg protein) were incubated at 37°C for 30 min with 25 μM trans-4-hydroxytamoxifen (trans-4-OH-TAM) or 15 μM cis-4-OH-TAM and 4 mM UDP-glucuronic acid prior to analysis by HPLC as described in Materials and methods. Propranolol was added to all assays postincubation as an internal standard for HPLC. (a) trans-4-OH-TAM incubated with UGT1A4-overexpressing microsomes; (b) cis-4-OH-TAM incubated with UGT1A4-overexpressing microsomes; (c) trans-4-OH-TAM incubated with UGT1A4-overexpressing microsomes and 1 N NaOH; (d) cis-4-OH-TAM incubated with HLM and 1 N NaOH. Peak 1, propranolol; peak 2, cis-4-OH-TAM; peak 3, trans-4-OH-TAM; peak 5, trans-4-OH-TAM-N
+-glucuonide; peak 7, cis-4-OH-TAM-N
+-glucuonide (peak numbers consistent with those described for Figure 1). mAU, milli-absorbance units.