High performance liquid chromatography determination of cytochrome P450 1A and 2C activities in bovine liver microsomes.
This study reports fluorescence high performance liquid chromatography (HPLC) and UV-Vis HPLC methods for the determination of 7-ethoxyresorufin O-deethylase (EROD) and tolbutamide methylhydroxylase (TMH) activities, respectively, using bovine liver microsomes. The detection limits were 0.022 and 5.5 pmol on the column, respectively; intra-day and inter-day precisions (expressed as relative standard deviation) were <10%. Both methods showed enough sensitivity to allow for an accurate determination of enzyme kinetic parameters according to Michaelis-Menten plots and the results were: K(m)=0.23+/-0.051 microM, V(max)=0.488+/-0.035 nmol/min/mg protein for EROD activity, and K(m)=1010+/-155.7 microM, V(max)=0.089+/-0.006 nmol/min/mg protein for TMH activity. An Eadie-Hofstee plot analysis showed that in bovine liver microsomes, EROD and TMH activities followed a monophasic kinetic pattern. alpha-Naphthoflavone, a cytochrome P450 1A1/2 (CYP1A1/2) inhibitor, and sulfaphenazole, a cytochrome P450 2C9 (CYP2C9) inhibitor, decreased EROD and TMH activities, respectively. The sensitivity of the methods allowed the use of microsomes with low enzyme activity, such as those from veal calf liver. Thus, EROD and TMH activities may be adopted as markers for the evaluation of CYP1A and CYP2C9-like activities in liver microsomes from veal and beef cattle.
Pegolo
Sara
S
Department of Public Health, Comparative Pathology and Veterinary Hygiene, University of Padua, Viale dell'Università 16, 35020 Legnaro, Padova, Italy.
Merlanti
Roberta
R
Department of Public Health, Comparative Pathology and Veterinary Hygiene, University of Padua, Viale dell'Università 16, 35020 Legnaro, Padova, Italy.
Giantin
Mery
M
Department of Public Health, Comparative Pathology and Veterinary Hygiene, University of Padua, Viale dell'Università 16, 35020 Legnaro, Padova, Italy.
Dacasto
Mauro
M
Department of Public Health, Comparative Pathology and Veterinary Hygiene, University of Padua, Viale dell'Università 16, 35020 Legnaro, Padova, Italy.
Montesissa
Clara
C
Department of Public Health, Comparative Pathology and Veterinary Hygiene, University of Padua, Viale dell'Università 16, 35020 Legnaro, Padova, Italy.
Capolongo
Francesca
F
Department of Public Health, Comparative Pathology and Veterinary Hygiene, University of Padua, Viale dell'Università 16, 35020 Legnaro, Padova, Italy. Electronic address: francesca.capolongo@unipd.it.
D000818:Animals; D000831:Animals, Newborn; D002138:Calibration; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods / Q000592:standards / Q000662:veterinary*; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013050:Spectrometry, Fluorescence / Q000662:veterinary
D003577:Cytochrome P-450 Enzyme System; D019363:Cytochrome P-450 CYP1A1; C053553:tolbutamide 4-hydroxylase