Measurement of cytochrome P450 2E1 activity in rat tracheobronchial airways using high-performance liquid chromatography with electrochemical detection

Cytochrome P450 2E1 catalyzes the metabolic activation of nitrosamines and haloalkenes to carcinogenic and cytotoxic derivatives; however, the regulation of this P450 isozyme is not well understood. Hydroxylation of p- nitrophenol to p-nitrocatechol has been used as a marker of CYP2E1 activity, but currently available methodologies are not sufficiently sensitive to allow measurements in small tissue samples or in tissues with low activity such as lung. We describe here a method for measuring p-nitrocatechol formation using HPLC with electrochemical detection which is rapid and specific. It has a level of sensitivity (pmol) sufficient to monitor CYP2E1 activities in incubations containing as little as 10 μg microsomal protein prepared from airway subcompartments of the lung, a tissue with low and varying CYP2E1 activities among different parts of the airways.