detected by mass spectrometry indicate that the phenyl group on the imidazole ring of SCH 66712 is one site of oxidation by CYP2D6 and could lead to methylene quinone formation. Three other metabolites were also observed. For understanding the metabolic pathway that leads to CYP2D6 inactivation, metabolism studies with CYP2C9 and CYP2C19 were performed because neither of these enzymes is significantly inhibited by SCH 66712. The metabolites formed by CYP2C9 and CYP2C19 are the same as those seen with CYP2D6, although in different abundance. Modeling studies with CYP2D6 revealed potential roles of various active site residues in the oxidation of SCH 66712 and inactivation of CYP2D6 and showed that the phenyl group of SCH 66712 is positioned at 2.2 Å from the heme iron.

Cytochrome P450 enzymes are a family of heme-containing proteins located throughout the body with roles in metabolism of endogenous and exogenous compounds. Among exogenous compounds, clinically relevant pharmaceutical agents are nearly all metabolized by P450 enzymes. However, the activity of the different cytochrome P450 enzymes varies among individuals and, therefore, so does drug efficacy as well as susceptibility to side effects and toxicity. Thus, assessing P450 activity is of great interest in drug development and clinical pharmacology. This study investigates the phenotyping of a single P450 activity by analyzing urine samples using isocratic reverse-phase HPLC. Specifically, the activity of human P450 1A2, which converts caffeine into paraxanthine, can be investigated by measuring the change in caffeine and paraxanthine concentrations in urine over time following a single dose of caffeine. There is an observable relationship between caffeine intake and paraxanthine formation that varies among individuals. This laboratory exercise provides a means for simple assessment of P450 1A2 metabolic activity using an HPLC method without additional extraction or purification steps and introduces students to the complexities of individualized medicine as well as the basic biochemical techniques of sample preparation and quantitative HPLC. Furthermore, students may design and test their own hypothesis using these methods.

Cytochrome P450 enzymes are a family of heme‐containing proteins located throughout the body with roles in metabolism of endogenous and exogenous compounds including nearly all pharmaceutical agents. The activity of the different cytochrome P450 enzymes varies among individuals and thus affects metabolism of foreign compounds. The goal of this study was to assess P450 1A2 metabolic activity by measuring caffeine and paraxanthine present in human urine following consumption of caffeine using isocratic reverse‐phase HPLC. Paraxanthine detected in all collected urine samples ranged in concentration from 1.93 μg/mL to 51.98 μg/mL. Caffeine was detected in most urine samples and ranged in concentration from 0.70 μg/mL to 77.17 μg/mL. The metabolic ratio of each urine sample at each time point was determined using the relationship MR = [paraxanthine]/[caffeine]. The range for MR values found was 0.34 and 26.34 and these values are within the ranges described in the literature by many groups and with much larger sample size. This laboratory exercise provides a means for simple assessment of P450 1A2 metabolic activity using a direct HPLC method and introduces students to the complexities of individualized medicine as well as the basic biochemical techniques of sample preparation and quantitative HPLC. (Supported in part by: Dreyfus Foundation, Kalamazoo College Kaufmann Fund, Michigan Economic Development Corporation).