Practical Tips

1. Incubations should be optimized with respect to incubation time and [E], preferably minimizing both.

2. Enzyme inactivation should be minimal during the incubation.

3. A pilot study should be performed using a wide range of substrate concentrations to estimate Km. This allows for identification of enzymes with low and/or high Km values.

4. For definitive study, a substrate concentration range from 3 x Km to Km/3 could be considered. Equal intervals of the difference between the inverse values of the outer substrate concentrations usually works well. For example, if Km is estimated at 5 mM, the concentration range would be from 15 to 1.67 mM. If six concentrations are used, then subtracting the inverse of the outer concentrations of the range from each other and dividing by six gives equal intervals of 0.0887

((1/1.67-1/15)/6 = 0.0887). This translates to [S] = 1.67, 2.37, 3.00, 4.10, 6.43, and 15.0 mM.

5. It is important that the consumption of the substrate is kept at minimum during the incubation. Ideally, consumption should be <10%, but practically this limit is usually <20% especially at lower concentrations, where sensitivity for detecting the substrate may become a limitation. A number of ways to overcome this obstacle are increasing incubation time, increasing [E], concentrating the samples before analysis, and monitoring metabolite formation which may be more sensitive.

6. In addition to monitoring substrate depletion, it is helpful to monitor the major metabolite(s) formed. Note that for Km determination, no absolute quantification of substrate or metabolite(s) is necessary as the peak area ratio is enough for initial evaluation.

Sometimes the metabolite formed is a more potent inhibitor of the enzyme than the substrate, thus complicating the interpretation of the kinetics. Ideally, testing the major metabolites for inhibitory properties is helpful, but not always possible.

See Table 5.2.

Table 5.2. In vitro probe reactions of major P450 isoforms

P450 isoform

Reaction (Km in |mM)

CYP1A2 Phenacetin O-deethylation (47a); tacrine 1-hydroxylation (3-16); 7-ethoxyresorufin O-deethylation (0.2-0.5); theophylline N-demethylation (200-600); caffeine-3 N-demethylation (150-600) CYP2A6 Coumarin 7-hydroxylation (0.84a)

CYP2B6 Bupropion hydroxylation (82a)

CYP2C8 Amodiaquine N-deethylation (1.9a); paclitaxel

6-a-hydroxylation (4-27) CYP2C9 Tolbutamide 4-methylhydroxylation (150a); diclofenac

4'-hydroxylation (4.0a) CYP2C19 (S)-Mephenytoin 4-hydroxylation (57a), omeprazole

5-hydroxylation (2-6) CYP2D6 Dextromethorphan O-demethylation (4.6a); bufuralol

1-hydroxylation (3-22) CYP2E1 Chlorzoxazone 6-hydroxylation (74a)

CYP3A Testosterone 6ß-hydroxylation (46a); midazolam-

1'-hydroxylation (2.3a)

aKm in human liver microsomes as reported by Walsky and Obach (2004) All other data are ranges of Km reported since 2000 in human liver microsomes

For determination of CYP3A inhibitory properties, two structurally diverse probe substrates are used (typically midazolam and testosterone), and the most potent result is used as an indicator of the drug's inhibitory property.

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