Interlaboratory comparison of microsomal ethoxyresorufin and pentoxyresorufin O-dealkylation determinations: standardization of assay conditions.
Assay conditions and results of cytochrome P-450 dependent 7-ethoxyresorufin (ER) and 7-pentoxyresorufin (PR) O-dealkylation (OD) by rat liver microsomes were compared by four laboratories in the Netherlands. Microsomal mixtures were prepared from control, 3-methylcholanthrene and phenobarbital pretreated animals, resulting in different levels of cytochrome P-450 isozymes. EROD and PROD activities were determined in each laboratory according to their own protocols. Considerable variability was found both between and within laboratories. Further studies demonstrated that protocol differences are important factors causing this interlaboratory variation. Main factors of influence were buffer type, batch of resorufin used for calibration, substrate solvent and substrate concentration. Based on the results obtained, standardized protocols for optimized measurement of microsomal EROD and PROD activities were developed. Additional experiments demonstrated that the use of these standardized protocols reduced intralaboratory variation in both the EROD and the PROD assay, whereas it also reduced the interlaboratory variability for the PROD determinations. The interlaboratory variation for measurement of microsomal EROD activities was only reduced for the laboratories using a Cobas-Bio analyzer. The results of the present study demonstrate clearly that data obtained with EROD and PROD activity measurements are highly sensitive to factors frequently varying from one laboratory to another. In addition, they demonstrate the necessity to be careful with absolute values presented in the literature for these activities, unless well characterized assay conditions are applied.
Rutten
A A
AA
TNO Toxicology and Nutrition Institute, Department of Biological Toxicology, Zeist, The Netherlands.
Falke
H E
HE
Catsburg
J F
JF
Wortelboer
H M
HM
Blaauboer
B J
BJ
Doorn
L
L
van Leeuwen
F X
FX
Theelen
R
R
Rietjens
I M
IM
D000818:Animals; D002021:Buffers; D002138:Calibration; D019363:Cytochrome P-450 CYP1A1; D019362:Cytochrome P-450 CYP2B1; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D003640:Dealkylation; D007753:Laboratories / Q000592:standards; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D010078:Oxazines / Q000378:metabolism / Q000494:pharmacology; D010088:Oxidoreductases / Q000032:analysis*; D011506:Proteins / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D015203:Reproducibility of Results; D012997:Solvents / Q000378:metabolism
D002021:Buffers; D010078:Oxazines; D011506:Proteins; D012997:Solvents; C007649:ethoxyresorufin; C014180:resorufin; C044943:pentoxyresorufin; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019363:Cytochrome P-450 CYP1A1; D019362:Cytochrome P-450 CYP2B1