Fast determination of low-level cytochrome P-450 1A1 activity by high-performance liquid chromatography with fluorescence or visible absorbance detection.
A method to determine the activity of the cytochrome P-450 1A1 enzyme, by measuring 7-ethoxyresorufin-O-deethylase (EROD) activity using high-performance liquid chromatography (HPLC) with fluorescence or with visible absorbance detection of resorufin, is described. The lowest quantifiable activity (0.2 pmol/mg min) is obtained by incubation of 0.3 mg of human duodenal microsomal proteins using HPLC fluorescence detection. Using HPLC with visible absorbance detection, sensivity was ten times lower. However, the equipment for this last method is available in most laboratories. The use of both HPLC assays allows determination of the low EROD activity level in samples of small size, such as two or three human duodenal biopsies obtained by routine endoscopy. These methods will be a useful tool to study the role of drug intestinal metabolism by cytochrome P-450 1A1.
Leclercq
I
I
Department of Gastroenterology, University Hospital St-Luc, Catholic University of Louvain, Brussels, Belgium.
Desager
J P
JP
Vandenplas
C
C
Horsmans
Y
Y
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019363:Cytochrome P-450 CYP1A1 / Q000032:analysis* / Q000378:metabolism; D004386:Duodenum / Q000201:enzymology / Q000648:ultrastructure; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology; D051381:Rats; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D019363:Cytochrome P-450 CYP1A1