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 ...Continue Reading
References
Aug 1, 1978·Proceedings of the National Academy of Sciences of the United States of America·Z H BegH B Brewer
Feb 1, 1990·Nature·J L Goldstein, M S Brown
Jul 1, 1973·Proceedings of the National Academy of Sciences of the United States of America·M S BrownJ L Goldstein
Dec 29, 1974·Biochimica Et Biophysica Acta·D J ShapiroR T Schimke
Jun 15, 1981·The Biochemical Journal·S PandianT S Raman
Apr 2, 1993·Journal of Chromatography·T YoshidaH Miyazaki
Dec 13, 2002·The Journal of Biological Chemistry·Sander M HoutenHans R Waterham
Jan 1, 2003·Clinical Chemistry·Patrick M BossuytUNKNOWN Standards for Reporting of Diagnostic Accuracy
Feb 1, 2003·Lipids·Marie-Yvonne Ndong-AkoumeIbrahim M Yousef
Jul 25, 2006·Journal of Lipid Research·G S SainiJ K Paliwal
Aug 1, 2006·FEBS Letters·Hans R Waterham
Feb 3, 2007·Journal of Lipid Research·Akira HondaHiroshi Miyazaki
Apr 29, 2009·Acta Paediatrica·Rolf Zetterström
Sep 2, 2009·Journal of Pharmaceutical and Biomedical Analysis·Joachim KuhnKnut Kleesiek
Oct 12, 2010·Journal of Lipid Research·Forbes D Porter, Gail E Herman
Nov 27, 2010·Biomedical Chromatography : BMC·Erick KindtScott T Fountain
Mar 1, 2011·Annals of Clinical Biochemistry·Jenna Waldron, Craig Webster
Oct 20, 2011·Cellular and Molecular Life Sciences : CMLS·Leigh Goedeke, Carlos Fernández-Hernando