High-performance liquid chromatographic assay for 4-nitrophenol hydroxylation, a putative cytochrome P-4502E1 activity, in human liver microsomes.
A high-performance liquid chromatographic method which measures formation of product 4-nitrocatechol (4NC) has been developed and applied to the study of human liver microsomal 4-nitrophenol (4NP) hydroxylation. Following diethyl ether extraction, 4NC and the assay internal standard (salicylamide) were separated by reversed-phase (C18) liquid chromatography. Extraction efficiencies of 4NC and internal standard were both > 90%. The assay, which has a limit of detection of 15 pmol injected (or an incubation 4NC concentration of 0.25 microM), is accurate, reproducible and straightforward. With a chromatographic time of 12 min, 40-50 samples may be analyzed per day. Rates of 4NC formation were linear with time and protein concentration to 50 min and 0.5 mg/ml, respectively. Preliminary studies of 4NP hydroxylation showed that this reaction followed single enzyme Michaelis-Menten kinetics in human liver microsomes.
Department of Clinical Pharmacology, Flinders Medical Centre, Bedford Park, Adelaide, Australia.
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W
Tassaneeyakul
M E
ME
Veronese
D J
DJ
Birkett
J O
JO
Miners
D002396:Catechols / Q000032:analysis / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005990:Glycerol / Q000494:pharmacology; D006801:Humans; D006900:Hydroxylation; D066298:In Vitro Techniques; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D009596:Nitrophenols / Q000032:analysis* / Q000378:metabolism / Q000493:pharmacokinetics; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D011786:Quality Control; D012997:Solvents; D013056:Spectrophotometry, Ultraviolet
D002396:Catechols; D009596:Nitrophenols; D012997:Solvents; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating; D005990:Glycerol; C001833:4-nitrocatechol; C024836:4-nitrophenol