Abstract
Radioisotope-based and mass spectrometry coupled to chromatographic techniques are the conventional methods for monitoring HMG-CoA reductase (HMGR) activity. Irrespective of offering adequate sensitivity, these methods are often cumbersome and time-consuming, requiring the handling of radiolabeled chemicals or elaborate ad-hoc derivatizing procedures. We propose a rapid and versatile reverse phase-HPLC method for assaying HMGR activity capable of monitoring the levels of both substrates (HMG-CoA and NADPH) and products (CoA, mevalonate, and NADP(+)) in a single 20 min run with no pretreatment required. The linear dynamic range was 10-26 pmol for HMG-CoA, 7-27 nmol for NADPH, 0.5-40 pmol for CoA and mevalonate, and 2-27 nmol for NADP(+), and limit of detection values were 2.67 pmol, 2.77 nmol, 0.27 pmol, and 1.3 nmol, respectively.
Highlights
Radioisotope-based and mass spectrometry coupled to chromatographic techniques are the conventional methods for monitoring HMG-CoA reductase (HMGR) activity
HMGR activity is conventionally assayed using elaborate radiochemical assay [4,5,6,7,8,9], chromatographic techniques coupled with mass spectrometry [10,11,12,13,14,15], or spectrophotometrically by monitoring the decrease in the absorbance of cofactor NADPH at 340 nm [16]
As an alternative for laboratories with no access to the expensive LC/MS equipment, we propose a rapid and adequately sensitive HPLC-based method capable of monitoring both the levels of all the species involved in the equilibrium in a single analysis and the kinetics of HMGR-catalyzed reactions
Summary
Radioisotope-based and mass spectrometry coupled to chromatographic techniques are the conventional methods for monitoring HMG-CoA reductase (HMGR) activity. As an alternative for laboratories with no access to the expensive LC/MS equipment, we propose a rapid and adequately sensitive HPLC-based method capable of monitoring both the levels of all the species involved in the equilibrium in a single analysis and the kinetics of HMGR-catalyzed reactions.
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