HPLC-fluorescence Determination of EROD Activity in Wistar Rat Liver Microsomes Obtained by Two Different Extraction Procedures

Katia      Roberta A. Belaz; Regina      V. Oliveira

Abstract

This work determines the total protein concentration present in microsome fractions obtained by two distinct extraction procedures: ultracentrifugation and calcium aggregation. Additionally, the batch to batch variability within the extraction procedures used and the microsome stability and activity at -80°C during 90 days were determined using 7- ethoxyresorufin as a marker substract. An HPLC method to determine the activity of the cytochrome CYP450 1A1/2 was developed and fully validated by measuring 7-ethoxyresorufin-O-deethylase (EROD) activity by in vitro microsomal mixed-function biotransformation. The biotransformation product, resorufin, was obtained by incubation of 500 μg of rat liver microsomal proteins. The incubation time was 5 min and the assay was monitored by using an HPLC coupled to a fluorescence detector (λexc 530 nm and λem 582 nm). The total analysis time was 20 min and the sample preparation was a fast and simple protein precipitation. The results show that both extraction procedures are useful tools for the extraction of subcellular fractions allowing the recovery of appropriate protein concentration for further in vitro metabolism studies by cytochrome P450, specifically CYP1A1 and CYP1A2.

Keywords: EROD activity, Calcium aggregation, Ultracentrifugation, Rat liver microsomes, HPLC-fluorescence, CYP1A1, CYP1A2, protein, ethoxyresorufin, microsomal