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-alkylprotoporphyrin IX (N-alkylPP), a potent inhibitor of ferrochelatase, the terminal enzyme in heme biosynthesis. Recognizing their role in experimental porphyria, 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 incubating porphyrinogenic compounds with rat liver microsomes. However, the overall 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 study we demonstrate that purified recombinant human ferrochelatase (FC) provides 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 this bioassay system might allow the use of human lymphoblastoid microsomal preparations 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 potentially porphyrinogenic drugs prior to administration to humans.
Department of Pharmacology and Toxicology, Faculty of Health Sciences, Queen's University, Kingston, ON, Canada.
J T
JT
Gamble
S G
SG
Wong
H A
HA
Dailey
G S
GS
Marks
D000818:Animals; D001681:Biological Assay; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005294:Ferrochelatase / Q000378:metabolism*; D006801:Humans; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D011524:Protoporphyrins / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D015262:Xenobiotics / Q000378:metabolism*
D011524:Protoporphyrins; D011994:Recombinant Proteins; D015262:Xenobiotics; D003577:Cytochrome P-450 Enzyme System; C028025:protoporphyrin IX; D005294:Ferrochelatase