purified enzyme systems to multienzyme (i.e., microsomes or hepatocytes)
systems. To utilize this approach, a standard probe substrate activity is used to
normalize expressed enzyme system activity to human liver microsomes. RAFs
are usually calculated as the ratio ofVmax(reaction velocity at saturating
substrate concentrations) in human liver microsomes of the enzyme-specific
index reaction divided by theVmaxof the reaction catalyzed by the cDNA-
expressed enzyme. Preferably the probe substrate activity should be conducted
in the same laboratory for both the expressed and microsomal systems. Once
the RAF for the enzyme of interest has been established using an index
substrate, the recombinant enzyme data can be converted to a microsomal
equivalent. Appropriate care should be used in the choice of probe substrate(s)
FIGURE 15.5 Definitive cytochrome P450 reaction phenotyping forN-demethylation
of sildenafil – (a) Use of selective enzyme inhibitors (b) Use of recombinant enzymes.
Reprinted from Hyland et al. (2001) by permission of Blackwell Publishing.
498 REACTION PHENOTYPING