Some in vitro assay conditions that affect detection and quantitation of phenobarbital-induced increases in hepatic microsomal drug-metabolizing enzyme activity

Abstract

Different laboratories use a variety of “standard” incubation conditions to assay in vitro for hepatic microsomal drug-metabolizing enzyme activity. Incubation volumes of 3–5 ml often contain 9000 g supernatant fractions or microsomes from 250 mg to 1.0 g of liver. Microsomal protein concentrations in such incubation mixtures will often exceed 2 mg/ml. Many workers do not specify rate of shaking or gas flow rate used in such incubations, and open air rather than flowing oxygen is often the gas phase. Under certain of these “standard” conditions we have been unable to demonstrate increased drug metabolism by hepatic fractions from phenobarbital-treated as compared with control rats. Incubation conditions contributing to this difficulty in demonstrating enzyme induction by phenobarbital were: (1) too much protein; (2) too slow shaking rate; (3) too little agitation of incubation mixtures; and (4) use of open air rather than flowing oxygen as gas phase. Incubation conditions which provided good evidence of the effects of phenobarbital as an inducer of the hepatic microsomal enzymes studied included: (1) microsomal protein concentrations in the incubation mixture of less than 2 mg/ml; (2) shaking rate of 100 oscillations per minute; (3) addition of a glass marble to the incubation mixture; and (4) gassing with oxygen rather than leaving open to the air.