title: Measurement of HMG CoA reductase activity in different human cell lines by ultra-performance liquid chromatography tandem mass spectrometry.
abstract: Hydroxymethylglutaryl coenzyme A reductase (HMGCR) catalyzes the rate limiting step in cholesterol biosynthesis converting HMG-CoA into mevalonic acid (MVA), which equilibrates with mevalonic acid lactone (MVL) under neutral pH conditions. We developed a fast, sensitive, and efficient method to determine HMGCR activity in human cell lines measuring MVL levels by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Convenient prepared samples containing MVL-D7 as an internal standard were injected, separated, and eluted from an ACQUITY HSS PFP column. Measurement of MVL was performed by electrospray ionization mass spectrometry with multiple reaction monitoring. Calibration curves were linear and reproducible in the range of 0.15-165 μg/l (r>0.99). Lower limit of quantification was 0.12 μg/l. Intra- and interassay imprecision were <1.3% and <2.9%, respectively. HMGCR enzymatic activity measurements of cells cultivated under different cell culture conditions (with 10% FCS, with 10% lipoprotein-deficient serum and under serum starvation) revealed the applicability of this test system for various experimental settings. This efficient UPLC-MS/MS assay permits rapid and high sensitive determination of HMGCR enzyme activity, tracing potential alterations in cholesterol biosynthesis.
authors: Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Georgstraße 11, 32 545 Bad Oeynhausen, Germany.
authors: Patricia
authors: P
authors: Kuzaj
authors: Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Georgstraße 11, 32 545 Bad Oeynhausen, Germany.
authors: Joachim
authors: J
authors: Kuhn
authors: Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Georgstraße 11, 32 545 Bad Oeynhausen, Germany.
authors: Isabel
authors: I
authors: Faust
authors: Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Georgstraße 11, 32 545 Bad Oeynhausen, Germany.
authors: Cornelius
authors: C
authors: Knabbe
authors: Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Georgstraße 11, 32 545 Bad Oeynhausen, Germany. Electronic address: dhendig@hdz-nrw.de.
authors: Doris
authors: D
authors: Hendig
mesh_terms: D000465:Algorithms; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004789:Enzyme Activation; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000032:analysis / Q000378:metabolism*; D013048:Specimen Handling / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D006903:Hydroxymethylglutaryl CoA Reductases
keywords: 3-hydroxy-3-methylglutaryl-CoA; Cell culture; Cholesterol biosynthesis; HMG-CoA; HMGCR; Hydroxymethylglutaryl coenzyme A reductase; LLOQ; MVA; MVL; Mevalonate; UPLC–MS/MS; hydroxymethylglutaryl coenzyme A reductase; lower limit of quantification; mevalonic acid; mevalonic acid lactone; ultra-performance liquid chromatography tandem mass spectrometry