Hydrochloric Acid → Phosphate

Participants

Reaction Conditions

Reaction Steps

Into 5 μl of a solution of the test compound in dimethylsulfoxide, 750 μl of a 1/15M phosphate buffer (pH 7.4) which was prepared by dissolving a wrapper of phosphate buffer powder (Yatron) in 10 ml of water, 40 μl of a 30 mM aqueous calcium chloride solution, 50 μl of a 20 mM aqueous GSH (glutathione, reduced form) solution, 50 μl of a 40 mM aqueous ATP (adenosine-5-triphosphate) solution and 100 μl of guinea pig polymorphonuclear leukocyte enzyme were added, followed by stirring at 25° C. for 5 minutes. To initiate the reaction, 5 μl of arachidic acid (10 mg/ml ethanol) were added to said mixture and the mixture was stirred at 25° C. for 5 minutes. 50 μl of 2N hydrochloric acid were added to the reaction mixture to terminate the reaction. 2.0 ml of ethyl acetate containing an internal standard substance (internal standard substance: 2 μg/ml of isoamyl paraben) were then added to the reaction mixture. The resulting mixture was extracted for 1 minute by a mixer, followed by centrifugal separation at 3000 rpm for 5 minutes. The ethyl acetate layer was separated, evaporated to dryness under reduced pressure and then dissolved in 200 μl of acetonitrile. By high-performance liquid chromatography, the amount of 5-HETE formed was determined. From the determination of 5-HETE of the control without the test compounds and that of the test compounds, an inhibition rate (%) was found and the 50% inhibition concentration of the test compounds against 5-lipoxygenase was determined. The results are shown in Table 12.

Provenance & Evidence