Indomethacin → 5-Hydroxyicosa-6,8,11,14-tetraenoic acid

Participants

Reaction Steps

For enzyme assay, 100-200 μl of enzyme was incubated in the presence and absence of test compound (prepared in DMSO) for 10 minutes at 30° C. in 0.05M Tris-HCl buffer (pH 7.2) containing 1 mM glutathione, 2 mM CaCl2, 14 μM indomethacin and 0.25 and 0.5 mM EDTA (final volume 400 μl). The final DMSO concentration in these assays was 4%. The assay was initiated by the addition of 100 μl 14C-arachidonic acid (58 mCi/mmole) and incubations were carried out for 10 minutes at 37° C. in a shaking water bath. The reactions were stopped by the simultaneous addition of 4.0 ml of diethylether and 100 μl of 1.0 m citric acid, while the tubes were vigorously vortexed. The 14C-5-HETE generated by the enzyme was extracted into the acidified ether layer which was separated from the aqueous layer by freezing the assay tubes in a dry ice/acetone bath prior to decanting the liquid ether layer. The ether layer was evaporated to dryness under N2, the residue dissolved in 65 μl chloroform:methanol (2:1 v/v), prior to separating 14C-5-HETE from other 14C-labeled compounds on silica gel impregnated glass fiber TLC plates utilizing an elution solvent of isoctane:methylethylketone:acetic acid (100:9:1 v/v). 14C-5-HETE was identified by its co-chromatography with a chemically synthesized standard. The radioactive peaks of the chromatogram were cut out and the radioactivity was quantitated by liquid scintillation counting. The amount of 5-HETE obtained per sample was expressed as the percent of total and was calculated in the basis of: ##EQU2## in order to evaluate the effect of the test compounds, the mean percent inhibition of 14C-5-HETE production was calculated. The IC50 for a drug was determined from a linear regression analysis of a plot of the mean percent inhibition vs drug concentration.

Provenance & Evidence