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 0.15–165μg/l (r>0.99). Lower limit of quantification was 0.12μg/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.
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