A Microassay for the detection of low levels of cytochrome P450 O-deethylation activities with alkoxyresorufin substrates

A microfluorometric method for the detection of low levels of cytochrome P450 was developed to increase the sensitivity of the assay, since a low level of CYP450 associated enzymatic activities was expected in human placenta tissues and small samples of placenta (∼10 g) could be easily collected, stored and processed. The dual fluorescence assay of Kennedy et al. [1], which was developed to simultaneously quantitate microsomal proteins and ethoxyresorufin-O-deethylase (EROD) activity was adapted for 96 wells microtiter plates. Placental microsomes samples were analyzed. For samples obtained from non-smoking mothers from the general southern Quebec population, results ranged from less than 1–3.3 pmol/mg protein•min. Samples collected from smoking mothers showed activity levels ranging from 30–69 pmol/mg protein•min. These results showed the suitability of the microassay for measuring low level of CYP450 activity in tissues such as placenta. (Mol Cell Biochem 175: 131–136, 1997)