TY - JOUR
T1 - High-performance liquid chromatographic assay for 4-nitrophenol hydroxylation, a putative cytochrome P-4502E1 activity, in human liver microsomes
AU - Tassaneeyakul, Wongwiwat
AU - Veronese, Maurice E.
AU - Birkett, Donald J.
AU - Miners, John O.
PY - 1993/6/23
Y1 - 1993/6/23
N2 - 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 μM), 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.
AB - 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 μM), 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.
UR - http://www.scopus.com/inward/record.url?scp=0027253618&partnerID=8YFLogxK
U2 - 10.1016/0378-4347(93)80473-H
DO - 10.1016/0378-4347(93)80473-H
M3 - Article
C2 - 8376495
AN - SCOPUS:0027253618
SN - 0378-4347
VL - 616
SP - 73
EP - 78
JO - Journal of Chromatography B: Biomedical Sciences and Applications
JF - Journal of Chromatography B: Biomedical Sciences and Applications
IS - 1
ER -