| Assay Method Information | |
| | LCAT Activity Measurement (In Vitro) |
| Description: | A fraction composed of HDL3 (1.125<specific gravity <1.210 g/mL) was obtained from the plasma of a healthy person by density gradient centrifugation. The obtained fraction was dialyzed against phosphate-buffered saline (pH 7.4) and used as an enzyme source and an acceptor for LCAT. Each test drug was prepared by dissolution in dimethyl sulfoxide. [14C]Cholesterol containing DTNB (Ellman's reagent, final concentration: 0.5 mM), mercaptoethanol (final concentration: 12.5 mM), and 0.6% bovine serum albumin was added to phosphate-buffered saline (pH 7.4) containing 1 mg/mL HDL3, and the test drug was further added thereto at varying concentrations to adjust the whole amount to 80 μL. This mixture was incubated at 37° C. for approximately 16 hours. Then, a mixed solution of hexane and isopropanol (mixing ratio=3:2) was added thereto to stop the reaction. After stirring, the hexane layer was collected, and this layer was evaporated to dryness. A chloroform solution (concentration: 10 mg/mL) was added thereto, and the mixture was spotted onto a thin-layer silica gel plate and developed using a mixed solution of hexane, diethyl ether, and ethyl acetate (mixing ratio=85:15:2). The radioactivity of a portion corresponding to cholesterol oleate was measured using an imaging analyzer BAS-2500 (manufactured by Fujifilm Corp.). A sample non-supplemented with the test drug was similarly treated and assayed. The EC50 value of LCAT activation was calculated according to the expression given below relative to the LCAT activity in the sample non-supplemented with the test drug. |
| Affinity data for this assay | |
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