A Comparison of Calcium Aggregation and Ultracentrifugation Methods for the Preparation of Rat Brain Microsomes for Drug Metabolism Studies.

ABSTRACT

Preparation of brain microsomes by the calcium chloride aggregation method has been suggested as an alternative to the ultracentrifugation method. However, the effects of the calcium chloride concentration on the quality of the microsomal fractions are not known. Brain microsomes were prepared from the adult rat brains using the high-speed ultracentrifugation and low-speed calcium chloride (10-100 mM) aggregation methods (n = 5-6 per group). The microsomal protein yield (spectrometry), the cytochrome P450 reductase (CPR) activity (spectrometry), and the monooxygenase activities (UPLC-MS/MS) of CYP2D and CYP2E1 were determined in the obtained fractions. Increasing the concentrations of calcium chloride progressively increased the protein yield of the low-speed microsomal fractions. However, the increased yield was associated with a significant decrease in the activities of CPR, CYP2D, and CYP2E1. Additionally, the CYP2D and CYP2E1 activities were significantly correlated with the CPR activities of the fractions. In conclusion, when an ultracentrifuge is available, preparation of brain microsomes by the ultracentrifugation method might be preferable. However, the calcium aggregation method at a calcium chloride concentration of 10 mM is an acceptable alternative to the ultracentrifuge method.