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Optimization, Validation and Application of Spectrophotometric Assay for 3-Hydroxy-3-methylglutarylcoenzyme A Reductase Activity
Abstract
Purpose: To improve the sensitivity and specificity of spectrophotometric 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase activity assay.
Methods: Spectrophotometric HMG-CoA reductase detection in male Wistar rat liver microsomes was optimized by applying different conditions, such as reaction buffer pH, NADPH and protein concentration, and preincubation and reaction times. The optimal set of conditions was validated using HMG-CoA reductase inhibitors, namely, pravastatin, fluvastatin, and rosuvastatin. IC50 was calculated and compared with that of a radiochemical assay. Ginkgo biloba extract’s (GBE50) inhibitory effect on HMG-CoA reductase activity was evaluated using the optimized spectrophotometric protocol.
Results: The optimum assay conditions were as follows: reaction buffer pH 7.0, 100 μM NADPH, 50 μM HMG-CoA, and 200 μg/mL microsomal protein. The preincubation and reaction times were 20 and 60 min, respectively, at 37 ºC. The IC50 of pravastatin, fluvastatin, and rosuvastatin under the optimum condition was 0.026, 0.015, and 0.007 μM while for radioisotope assay, it was 0.034, 0.049 and 0.0119 μM, respectively. GBE50 significantly inhibited HMG-CoA reductase activity in a concentrationdependent manner (p < 0.05).
Conclusion: These results suggest that HMG-CoA reductase activity can be detected using the improved spectrophotometric assay. This assay can facilitate the discovery and development of new HMG-CoA reductase inhibitors in vitro.
Keywords: Spectrophotometry, 3-Hydroxy-3-methylglutaryl-coenzyme A reductase activity, Cholesterol metabolism, Ginkgo biloba, Pravastatin, Fluvastatin, Rosuvastatin
