Spectrophotometric measurement of flavin-containing monooxygenase activity in freshly isolated rat hepatocytes and their cultures.
The determination of the mixed function flavin-containing monooxygenase activity in rat liver and in hepatocytes and their cultures by spectrophotometric measurement of the oxygenation of methimazole is complicated by an inhibition caused by some of the reagents used during this method. Optimal conditions were determined for measuring this enzyme activity in microsomal preparations of rat liver and its hepatocytes. Optimal flavin-containing monooxygenase activities were obtained for measurements performed in a 0.25 M N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine-EDTA buffer at pH 8.7 and at a methimazole concentration of 2 mM. Data are also presented which show that no interferences caused by either cytochrome P450-dependent enzymes or by the reduction of methimazole disulfide by glutathione have to be taken into account when determining methimazole oxygenation. Finally, the above assay was also used to study flavin-containing monooxygenase activity in primary monolayer cultures of hepatocytes for 6 days.
Coecke
S
S
Department of Toxicology, Vrije Universiteit Brussel, Belgium.
Mertens
K
K
Segaert
A
A
Callaerts
A
A
Vercruysse
A
A
Rogiers
V
V
D000818:Animals; D002478:Cells, Cultured; D004126:Dimethylformamide / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D010105:Oxygenases / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013053:Spectrophotometry / Q000379:methods
D004126:Dimethylformamide; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); D005033:Ethylmaleimide