Use of recombinant human ferrochelatase as a sensitive bioassay for N-alkylprotoporphyrin IX formed after interaction of porphyrinogenic xenobiotics with rat liver microsomes

Several porphyrinogenic xenobiotics elicit mechanism-based inactivation of cytochrome P450 (CYP) isozymes, leading to the formation of N-alkylprotopor phyrin IX (N-alkylPP), a potent inhibitor of ferrochelatase, the terminal e nzyme in heme biosynthesis. Recognizing their role in experimental porphyri a, our long term objective is the establishment of an appropriate in vitro system for the detection and quantification of N-alkylPPs, formed in human liver after the administration of potential porphyrinogenic compounds. In a previous study, we used a combination of thin-layer chromatography and UV- visible spectrophotometry to isolate and identify N-alkylPPs after incubati ng porphyrinogenic compounds with rat liver microsomes. However, the overal l yield of N-alkylPPs was low, and it was concluded that in vitro systems, such as human lymphoblastoid microsomal preparations containing single cDNA -expressed human cytochrome P450 (CYP) isozymes, do not contain sufficient CYP for in vitro studies designed to isolate N-alkylPP. In the present stud y we demonstrate that purified recombinant human ferrochelatase (FC) provid es an extremely sensitive bioassay system for N-alkylPPs and is capable of detecting N-alkylPP in the 10(-6) nmol range. Therefore, we propose that th is bioassay system might allow the use of human lymphoblastoid microsomal p reparations containing single cDNA-expressed human CYP isozymes to detect N -alkylPP produced after mechanism-based (catalysis-based) CYP inactivation. If this is found to be correct it will facilitate identification of potent ially porphyrinogenic drugs prior to administration to humans.