Interlaboratory comparison of total cytochrome P-450 and protein determinations in rat liver microsomes
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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.
Key wordsCytochrome P-450 determination Protein determination Liver microsomes Interlaboratory comparison
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- Bartosek I, Dolfini E, Ghersa P, Guaitani A, Villa P, Villa S (1980) Preservation of rat hepatic microsomal enzyme activities: Effect of low temperature and freeze-drying. J Pharmacol Methods 3: 191–200Google Scholar
- Danner-Rabovsky J, Groseclose RD (1982) Stability of rat lung and liver microsomal cytochrome P-450 enzyme activities to storage: purified microsomal fraction, postmitochondrial fraction, and whole tissue. J Toxicol Environ Health 10: 601–611Google Scholar
- Dent JG, Schnell SS, Graichen ME, Allen P, Abernethy D, Couch DB (1981) Stability of activating systems for in vitro mutagenesis assays: Enzyme activity and activating ability following long-term storage at -80° C. Environ Mutagen 3: 167–179Google Scholar
- Hayes AW (1982) Principles and methods in toxicology. Raven Press, New York, pp 611–612Google Scholar
- Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with Folin phenol reagent. J Biol Chem 193: 265–275Google Scholar
- Matsubara T, Koike M, Touchi A, Tochino Y, Sugeno K (1976) Quantitative determination of cytochrome P-450 in rat liver homogenate. Anal Biochem 75: 596–603Google Scholar
- Omura T, Sato R (1964a) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chem 239: 2370–2378Google Scholar
- Omura T, Sato R (1964b) The carbon monoxide-binding pigment of liver microsomes. II. Solubilization, purification, and properties. J Biol Chem 239: 2379–2385Google Scholar
- Pearson K (1981) In: Sokol RR, Rohlf FJ (eds) Biometry, 2nd ed. W. H. Freeman and Company, New York, pp 565–601Google Scholar
- Schoene B, Fleischman RA, Remmer H, v. Oldershausen HF (1972) Determination of drug metabolizing enzymes in needle biopsies of human liver. Eur J Clin Pharmacol 4: 65–73Google Scholar