Cytochrome P-450 measurement in rat liver homogenate and microsomes. Its use for correction of microsomal losses incurred by differential centrifugation

Abstract

Cytochrome P-450 was assayed in rat liver homogenates and microsomes in order to calculate microsomal recoveries and correct for losses during ultracentrifugation or sedimentation in presence of CaCl2. The values obtained for corrected microsomal protein in untreated female Sprague-Dawley rats were between 40 and 50 mg/g of liver. The assay of cytochrome P-450 in liver homogenate is accurate enough to calculate a reproducible recovery factor. The value of the method lies in its rapidity, its capacity to correct over a wide range of losses, and its capacity to yield reliable values of the total microsomal protein mass. The limits of this method include overestimation of homogenate cytochrome P-450 and inability to correct for nonmicrosomal protein contamination. Overestimation of cytochrome P-450 can be corrected by measuring the difference in absorbance between 450 and 510 nm with the extinction coefficient of 100 mM-1cm-1. To be accurate, cytochrome P-450 determination on microsomes must be done at protein concentrations of about 3 mg/ml. The error inherent to the method may be kept constant and minimal. The use of correction for microsomal losses is recommended in order to obtain uniformity between results from various laboratories and adequate correlation with in vivo studies of microsomal functions.