15.3 Noncytochrome P450 Reaction Phenotyping
Similar to P450 reaction phenotyping, designing experimental studies to
elucidate the contribution of non-P450 enzymes requires an understanding
of the particular enzymes substrate specificity, mechanism of reaction catalysis,
tissue expression level, subcellular location, required cofactors/experimental
conditions, and selective inhibitors (if identified). The following sections will
focus on several examples of non-P450 reaction phenotyping including flavin-
containing monooxygenases (FMOs), monoamine oxidases, and esterases.
15.3.1 Flavin-Containing Monooxygenases
Human flavin-containing monooxygenases are NADPH dependent micro-
somal enzymes that catalyze the oxygenation of many nitrogen, sulfur,
selenium, and phosphorous heteroatom-containing chemicals and drugs
(Cashman, 1995, 2003; Ziegler, 1993). However, in the top 200 drugs prescribed
in the United States, FMOs account for only a small portion of the metabolic
pathways when compared to P450s and UDP-glucuronosyltransferases
(UGTs) (Williams et al., 2004). Currently there are five functional human
FMOs known (Lawton et al., 1994) and FMO3 appears to be the most
important FMO present in adult human liver. Based on immunoreactivity
studies (Overby et al., 1997), FMO3 is present in adult human liver (Cashman,
2004), in contrast to FMO1 which is not present to any extent in adult liver.
However, the expression levels are reversed in fetal livers where FMO1
predominates and FMO3 is not detected (Cashman, 2004; Koukouritaki et al.,
2002; Yeung et al., 2000). FMO1 is also located in adult human kidney (Yeung
et al., 2000). FMO5 is located in human liver, however, it is more substrate
limited than FMO3 (Cashman, 2002; Overby et al., 1995). FMO1, FMO3, and
FMO5 are currently available commercially as expressed enzymes. Since FMOs
are oxidative enzymes that share a subcellular fraction (microsomes) and
cofactor (NADPH) with cytochrome P450 enzymes, both systems will be active
in native microsomes under common experimental conditions. Thus,
differentiation of FMO and P450 activity is achieved either by selectively
inactivating the P450 or the FMO enzymes. To inactivate the P450 component,
a pan P450 inhibitor (i.e., aminobenzotriazole) or a detergent can be utilized (as
P450 enzymes are more sensitive than FMO enzymes to surfactants). To
inactivate the FMO component, either chemical inhibition (i.e., methimazole)
or thermal degradation can be used. In comparison to cytochrome P450s,
FMOs are less thermally stable and can be inactivated by preincubating for
5 min at 45C in the absence of NADPH (prior to cooling to 37C and initiating
the reaction). Since FMO is thermolabile microsomal preparation technique
can impact FMO activity, therefore when studying FMO dependent
metabolism it is important to use well-characterized microsomes (i.e.,
microsomes identified to have high functional FMO activity using a probe
substrate).
NONCYTOCHROME P450 REACTION PHENOTYPING 481