Table 1.
Summary of the reaction conditions and estimated Michaelis-Menten parameters of the CYP isoform-selective marker reactions
| CYP | Substrate/solvent Inhibitor/solvent | Substrate concentration in assay (range) (µm)a | Incubation time(min) | Quenching method | Km1 (Km2)(µm)b | Vmax1 (Vmax2)(pmol mg−1 min−1)b |
|---|---|---|---|---|---|---|
| CYP1A2 | Phenacetin/methanol Furafylline/methanol | 10 (5–250) | 30 | 100 µl acetonitrile | 19 (556) | 836 (824) |
| CYP2A6 | Coumarin/methanol NAc | 0.5, 1, 5 (0.1–50) | 10 | 20 µl 70% perchloric acid | 1.1 | 1303 |
| CYP2C9 | Tolbutamide/ethanol Sulfaphenazole/methanol | 25, 50, 100, 250 (10–250) | 60 | 20 µl 85% phosphoric acid | 65 | 194 |
| CYP2C19 | S-mephenytoin/methanol Tranylcypromine/water | 25, 50, 75, 100 (10–250) | 60 | 20 µl 85% phosphoric acid | 39 | 102 |
| CYP2D6 | Dextromethorphan/water Quinidine/methanol | 1.5 (0.5–50) | 20 | 20 µl 70% perchloric acid | 1.5 | 227 |
| CYP2E1 | Chlorzoxazone/methanol Pyridine/water | 30 (5–250) | 30 | 20 µl 85% phosphoric acid | 27 | 807 |
| CYP3A4 | Midazolam/acetonitrile Troleandomycin/methanol | 2, 5, 10, 15 (0.5–50) | 6 | 200 µl methanol | 1.4 | 1858 |
a
Concentrations used in the inhibition study were chosen around appropriate apparent Km values. Values in parenthesis are the substrate concentration ranges used for characterization of Km and Vmax.
b
A two-enzyme Michaelis-Menten equation was best fitted to data for phenacetin O-deethylation (the apparent kinetic data for CYP1A2 are shown without parentheses), in agreement with previous studies. A single Michaelis-Menten equation was best fitted to data for the other marker reactions.
c
Chemical (8-methoxypsoralen) not available.