Assay Conditions for Diclofenac 4'-Hydroxylation

Diclofenac 4’-hydroxylation is a low K_m, high turnover assay for human CYP2C9. The apparent K_m for the CYP2C9-catalyzed reaction is 2-3 uM. The turnover number (per unit cDNA-expressed CYP2C9*1 is over 40 min^-1). When performing kinetic analyses near the apparent K_m, protein concentration and incubation time should be chosen to avoid excessive metabolism of substrate. GENTEST supplies 4’-hydroxydiclofenac metabolite standard (Catalog. No. 451443 (Old B443)) for quantitation of the assay results.

Solutions
I.      0.4 mM Diclofenac in 0.1 M Tris pH 7.5
II.     20 mg/ml Glucose 6-phosphate, 20 mg/ml NADP, 13.3 mg/ml MgCl2-H2O
III.    40 U/ml Glucose 6-phosphate dehydrogenase in 5 mM sodium citrate (tribasic)
IV.    0.1 M Tris pH 7.5
V.     94% Acetonitrile, 6% glacial acetic acid

Incubation Conditions (for 1 ml Final Volume):
50 ul           Solution II.
10 ul           Solution III.
250 ul         Solution I. (Final concentration 100 uM, a saturating concentration)
xx ul           Enzyme. (human liver microsomes or cDNA-expressed)
690 - xx ul  Solution IV.

Mix and prewarm to 37^oC all solutions except enzyme. Initiate incubation with the addition of enzyme. After the desired incubation time, stop the reaction by the addition of 200 ul of solution V and cool on ice. Centrifuge 12000 x g for 4 minutes to precipitate protein. Analyze the supernatant for product formation by HPLC separation with UV detection. Recommended range of injection volumes - 10 to 150 ul.

HPLC Conditions
Mobile Phase A: 30% Acetonitrile, 70% water, 1 mM Perchloric acid (See Note 1)
Mobile Phase B: 100% Methanol
Gradient: Initial conditions: 30% B with a linear gradient to 100% B over 20 minutes
Column: Nucleosil C18, 4.6 x 250 mm, 5 um particle size (see Note 2)
Temperature: 50^oC (see Note 3)
Flow Rate: 1 ml/min
Detector: Absorbance at 280 nm
Retention Times: 4’-Hydroxydiclofenac, 11 minutes; Diclofenac, 15 minutes

Note 1
Diclofenac and 4’-hydroxydiclofenac are being chromatographed at acidic pH with the carboxylic acid protonated. Separation can also be achieved at slightly alkaline pH (pH 7.4) when the carboxylic acid is ionized, however, column life may be adversely affected. This is the same mobile phase used for the bufuralol 1’-hydroxylase assay (GENTEST Catalog No. B311).
Note 2
4’-hydroxydiclofenac and diclofenac are easily separated and most C18 columns should be adequate for the purpose. However, some adjustment in mobile phase gradient conditions may be desired.
Note 3
Column temperature can range from room temperature to 50^oC. The use of a controlled, elevated temperature provides greater reproducibility in retention times and lower column back pressures.

References
J. Chromatography (1985) 338, 151; J. Chromatography (1990) 528, 487; Life Sciences (1993) 52, 29; J. Chromatography (1993) 620, 158.