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Originally published In Press as doi:10.1194/jlr.D600049-JLR200 on February 1, 2007

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Journal of Lipid Research, Vol. 48, 1212-1220, May 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Methods

Highly sensitive assay of HMG-CoA reductase activity by LC-ESI-MS/MS

Akira Honda1,*,{dagger}, Yuji Mizokami*, Yasushi Matsuzaki*, Tadashi Ikegami*, Mikio Doy{dagger} and Hiroshi Miyazaki§

* Department of Internal Medicine, Tokyo Medical University, Kasumigaura Hospital, Ami, Ibaraki 300-0395, Japan
{dagger} Ibaraki Prefectural Institute of Public Health, Mito, Ibaraki 310-0852, Japan
§ Pharmax Institute, Kawasaki, Kanagawa 213-0021, 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), ~833 times more sensitive than that of MVL measured by a conventional radioisotope (RI) method (200 fmol). The variances between sample preparations and between measurements by this method were analyzed by one-way layout and calculated to be 3.2% and 1.8%, respectively. The recovery experiments were performed using incubation mixtures spiked with 0.77–2.31 nmol MVL/mg protein and were validated by a polynomial equation. These results showed that the estimated concentration within a 95% confidence limit was 0.47 ± 0.07 nmol/mg protein, which coincided completely with the observed X0 nmol/mg protein with a mean recovery of 94.6%. This method made it possible to measure HMG-CoA reductase activity with a high degree of reproducibility and reliability, and especially with sensitivity superior to that of the conventional RI method.

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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Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.