The in vitro biological test system selected to metabolize ESL
was the human liver microsome system. The reaction was performed
by adding 192.6 l of 0.1 M phosphate buffer (pH 7.4) and
7.4 l of ESL (1 mg/ml) to a mixture of 25 l microsomes (diluted to
1 mg/ml with 0.1 M phosphate buffer) and 25 l of 5 mM NADPH in
order to reach a final substrate concentration of 100 M. The incubations
were performed at 37 ◦C in a ThermoMixer C (Eppendorf,
Hamburg, Germany) under gentle agitation (300 rpm). The reaction
was stopped after an incubation time of 60 min by immersion
of the sample in an ice bath for 10 min. Control samples were also
prepared. These samples were prepared by addition of phosphoric
acid solution (5%, m/v) to the microsomes.
2.6