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Journal of Lipid Research, Vol. 48, 1212-1220, May 2007
Highly sensitive assay of HMG-CoA reductase activity by LC-ESI-MS/MS
* Department of Internal Medicine, Tokyo Medical University, Kasumigaura Hospital, Ami, Ibaraki 300-0395, Japan Published, JLR Papers in Press, February 1, 2007.
1 To whom correspondence should be addressed. e-mail: akirahonda-gi{at}umin.ac.jp
We have developed a new sensitive and specific nonradioisotope assay method to measure the activity of HMG-CoA reductase, the rate-controlling enzyme in the cholesterol biosynthetic pathway. This method was based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry using electrospray ionization in positive mode. Mevalonic acid, the product of HMG-CoA reductase, was converted to mevalonolactone (MVL) in an incubation mixture, extracted by a salting-out procedure, derivatized into the mevalonyl-(2-pyrrolidin-1-yl-ethyl)-amide, and then purified using a disposable silica cartridge. The resulting mevalonylamide was quantified by selected reaction monitoring using the positive electrospray ionization mode. The detection limit of this mevalonylamide was found to be 240 amol (signal-to-noise ratio = 3),
Supplementary key words cholesterol biosynthesis mevalonic acid mevalonolactone liquid chromatography-electrospray ionization-tandem mass spectrometry 3-hydroxy-3-methylglutaryl-coenzyme A reductase Abbreviations: CI, chemical ionization; EI, electron ionization; GC, gas chromatography; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; MVA, mevalonic acid; MVL, mevalonolactone; MV-PLEA, mevalonyl-(2-pyrrolidin-1-yl-ethyl)-amide; P-ESI, electrospray ionization in positive mode; RI, radioisotope; S/N, signal-to-noise ratio; SREBP, sterol-regulatory element binding protein; SRM, selected reaction monitoring
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