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.

Interlaboratory comparison of total cytochrome P-450 and protein determinations in rat liver microsomes. Reinvestigation of assay conditions.

Assay conditions in determining total cytochrome P-450 in four laboratories were compared. Although the determination was derived from the original Omura and Sato method in each laboratory, the four standard protocols differed slightly, resulting in considerable differences in the results. Since the cytochrome P-450 content is usually expressed per mg protein, the protein assay conditions were evaluated as well. Furthermore, we compared the cytochrome P-450 values obtained by the CO- and the dithionite (DT)-difference methods. The effect of a number of variables in the assay was investigated. The influence of the storage temperature of the microsomes was ascertained as well as effects of the gassing time with CO and the time between addition of dithionite, CO-gassing and the recording of the difference spectra. After evaluating these variables a standard operation procedure was established. Using this procedure the interlaboratory coefficient of variation for total cytochrome P-450 was 4.8%, a value which was comparable to the intralaboratory coefficients of variation. The final results also show that the millimolar extinction coefficient for the DT-difference method is higher than for the CO-difference method.