Multiple FMO-3 variants have been identified. The best studied relationship
between FMO-3 polymorphisms and enzyme function is the association
between FMO3 genetic variants and trimethylamine (TMA) metabolism
(resulting in fish odor syndrome) (Cashman, 2002; Cashman and Zhang,
2002; Hernandez et al., 2003). Currently, FMO-3 (similar to the P450s) can be
evaluated utilizing expressed enzyme, correlation analysis, and controlled
incubation conditions (i.e., cofactor, chemical inhibitors, and/or thermal
degradation). Examples of FMO reaction phenotyping studies include the
evaluation of the enzyme system(s) responsible for theN-oxidation of clozapine
(Tugnait et al., 1997) and the metabolism of sulfinpyrazone sulfide and
sulfinpyrazone (He et al., 2001).
The involvement of FMO in the formation of theN-oxide of clozapine was
determined using chemical inhibition (FMO inhibitor: methimazole), heat
inactivation and protection against heat inactivation in the presence of
NADPH. In addition clozapineN-oxide was catalyzed by purified FMO3 with
aKmsimilar to that determined using human liver microsomes. In contrast,
sulfinpyrazone sulfide and sulfinpyrazone metabolism was determined to be
catalyzed by P450s rather than FMO using modified microsomal incubation
methods (heat inactivation and surfactant), specific P450 inhibitors with
microsomes, and expressed P450s.
15.3.2 Monoamine Oxidases A and B (MAO-A and MAO-B)
Monoamine oxidases are integral outer mitochondrial membrane proteins that
catalyze the oxidative deamination of primary and secondary amines as well as
some tertiary amines. MAO occurs as two enzymes, MAO-A and MAO-B,
which differ in substrate selectivity and inhibitor sensitivity (Abell and Kwan,
2001; Edmondson et al., 2004; Shih et al., 1999). A number of MAO inhibitors
have been developed for clinical use as antidepressants and as neuroprotective
drugs. Clinically used drug substances include, among others, moclobemide, a
relatively selective reversible MAO-A inhibitor, andL-deprenyl, an irreversible
selective inhibitor of MAO-B.In vitro, clorgyline andL-deprenyl are used as
selective irreversible inhibitors of MAO-A and B, respectively. (Note: Forin
vitro studies using irreversible inhibitors, preincubation of the irreversible
inhibitor with the enzyme prior to initiation of the substrate reaction is
required for optimal inhibition.) Expressed MAO-A and MAO-B are not
readily available via commercial resources; however, MAO-A and MAO-B
have been evaluated and are active in subcellular fractions. While monoamine
oxidases are located in the mitochondria, many microsomal preparations are
contaminated with monoamine oxidases during the preparation of the
microsomal subcellular fraction and thus microsomes are sometimes used to
evaluate monoamine oxidase activity in combination with selective inhibitors.
A study comparing monoamine oxidase activity between Japanese and
Caucasian livers used rizatriptan as the model substrate (Iwasa et al., 2003).
The Japanese livers were evaluated for monoamine oxidase activity using both
482 REACTION PHENOTYPING