Hydroxylation of the thiophene ring by hepatic monooxygenases. Evidence for 5-hydroxylation of 2-aroylthiophenes as a general metabolic pathway using a simple UV-visible assay.
The 5-hydroxylation of tienilic acid by rat liver microsomes was measured by a new, simple method involving the detection of 5-hydroxytienilic acid by UV-visible spectroscopy. This assay allowed continuous detection of this metabolite and could be easily used to determine the kinetic parameters of the reaction (Vmax and Km being respectively 1 +/- 0.2 nmol product formed/mg protein/min and 14 +/- 2 microM for liver microsomes from phenobarbital-treated rats). This activity was found to be dependent on NADPH and to be inhibited by CO, SKF 525A and metyrapone, indicating that it is dependent on cytochromes P-450. This UV-visible assay is based on intrinsic properties of 5-hydroxy 2-aroylthiophenes which exist as highly conjugated anions at physiological pH and exhibit large epsilon values around 390 nm. Its application to other 2-aroylthiophenes like suprofen, 2-parachlorobenzoylthiophene and a series of 2-aroylthiophenes with various substituents on the aroyl group showed that, in general, thiophene compounds bearing a 2-arylketo substituent appear to be hydroxylated at position 5 by rat liver microsomes. The kinetic parameters of the 5-hydroxylation of suprofen and 2-parachlorobenzoylthiophene by liver microsomes from phenobarbital-treated rats were determined and found to be similar to those for tienilic acid hydroxylation.
Neau
E
E
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, CNRS-INSERM, Université René Descartes, Paris, France.
Dansette
P M
PM
Andronik
V
V
Mansuy
D
D
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006900:Hydroxylation; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology* / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013876:Thiophenes / Q000378:metabolism*; D013989:Ticrynafen / Q000031:analogs & derivatives / Q000378:metabolism
D013876:Thiophenes; D003577:Cytochrome P-450 Enzyme System; C041405:5-hydroxythienilic acid; D010105:Oxygenases; D013989:Ticrynafen