CYP3A2 Activity

The activity of CYP3A2 was measured using erythromycin N-demethylation (63,64). Samples were prepared by mixing 1.0 mg of S9 protein, 0.4 mM erythromycin, and 1.0 mM ß-NADPH in a final volume of 1 mL in 0.1 M potassium phosphate buffer (pH 7.4). The samples were incubated for 15 min at 37°C, mixed with 0.5 mL of 17% perchloric acid (Sigma, St. Louis, MO), and centri-fuged at 15,000g for 5 min. Formaldehyde was measured by the colorimetric method of Nash (65). The samples were placed in a new tube and mixed with 0.4 mL of Nash reagent (0.02 M 2,4-pentanedione, 0.6% [v/v] glacial acetic acid, and 3.9 M ammonium acetate) and incubated at 70°C for 20 min. The final product was read on a spectrophotometer at 412 nm. Absorbance was compared to a standard curve generated from known concentrations of formal-

Fig. 3. Representative chromatogram depicting quantitative analysis of paclitaxel in plasma (see Subheading 3.4. for details).

dehyde. Activities were recorded as micromoles of formaldehyde per milligram of S9 protein per minute.

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